Combining forced limb use with Ninjin'yoeito treatment prevents atrophy in fast-twitch muscles and promotes functional restoration after hemorrhagic stroke in rat models

Combining forced limb use with Ninjin'yoeito treatment prevents atrophy in fast-twitch muscles and promotes functional restoration after hemorrhagic stroke in rat models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Combining forced limb use with Ninjin'yoeito treatment prevents atrophy in fast-twitch muscles and promotes functional restoration after hemorrhagic stroke in rat models Naoki Tajiri, Shinya Ueno, Dewi Mustika, Shiori Tominaga, Takeshi Shimizu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5964788/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rehabilitative training incorporating forced limb use (FLU) following intracerebral hemorrhagic stroke (ICH) enhances functional recovery of skilled reaching in rats. Given that Ninjin'yoeito (NYT) influences both cerebral and muscular systems, this study aimed to investigate whether the combined application of FLU and NYT could yield superior functional recovery compared to FLU alone. The ICH model was established by collagenase injection, and the subject was administered FLU from day 1 after ICH (D1) for 7 days and 1% NYT chow until D56. The combination of FLU and NYT resulted in significantly enhanced functional recovery in motor deficit scores at D28 and D56 compared with ICH only, although the score was comparable to that of the FLU group. The combination group exhibited increased total walking distance and a higher number of center entrances in the open-field test at D28. Retrograde labeling of corticospinal neurons after ICH with FluoroGold (FG) revealed no significant increase in FG-positive cells in the cortex of the combination group compared to the FLU group. Anterograde labeling with biotinylated dextran amine demonstrated increased bouton-like varicosities in the red nucleus, similar to that in the FLU group, although NYT alone did not increase the number of positive cells. Specific atrophy of MHC IIb-positive muscles after ICH was mitigated in the combination group, although no significant changes were observed in either the FLU or NYT groups. These findings indicate that the combination of FLU and NYT contributes to the functional recovery of FLU following ICH, mitigating atrophy of fast-twitch muscles. Intracerebral hemorrhage Rehabilitation Kampo medicines Functional recovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hemorrhagic stroke causes impairment of motor function, leading to difficulties in daily life [ 1 ]. Intracerebral hemorrhage (ICH) in proximity to the internal capsule results in relatively severe motor dysfunction, even in cases of small hemorrhage [ 2 – 5 ]. Post-stroke rehabilitation is considered an essential strategy for mitigating motor function impairment and enhancing patients' quality of life [ 6 ]. Intensive utilization of paralyzed limbs, such as constraint-induced movement therapy, is available for effective rehabilitation [ 7 – 9 ]. The effects of rehabilitative training on the injured brain are reported to induce neurophysiological and neuroanatomical plasticity, facilitating the formation of compensatory neuronal circuits and enhanced synaptic plasticity [ 10 , 11 ]. We have previously reported that intensive rehabilitative training of forced limb use (FLU) results in functional recovery in hemorrhagic stroke in rats, elucidating a causal link between the cortico-rubral pathway and its functional recovery [ 12 ]. A dynamic compensatory action to the cortico-brainstem pathways was also reported in FLU after ICH under both blockade of the corticospinal tract and the cortico-rubral tract [ 13 ]. In addition to rehabilitative training after stroke, the significance of Kampo medicine has progressively been acknowledged in stroke research [ 14 ]. Ninjin'yoeito (NYT) is a traditional Japanese Kampo medicine that affects the brain and muscle. Notably, the component crude-drugs of NYT, such as Rehmannia Root, Japanese Angelica Root, Peony Root, and Cnidium rhizome, have been demonstrated to reduce the infarct volume in ischemic stroke rats [ 15 ]. From a clinical perspective, NYT offers various benefits, including alleviation of fatigue, malaise, anorexia, frailty, sarcopenia, and cognitive dysfunction [ 16 – 25 ]. Furthermore, NYT enhances motivation [ 25 ], augments dopamine signaling [ 26 , 27 ], promotes remyelination [ 28 ], and facilitates the synthesis and secretion of nerve growth factor (NGF) [ 29 , 30 ]. Although interest in Kampo medicine for rehabilitative training is increasing [ 14 ], it remains unclear whether NYT itself has an effect on hemorrhagic stroke and whether NYT has an additive effect on rehabilitative training after ICH. In this investigation, we initially ascertained whether the administration of NYT augments the effects of rehabilitative training by FLU following hemorrhagic stroke, using an ICH rat model exhibiting relatively severe motor dysfunction even with small hemorrhages. Subsequently, we examined the underlying mechanism by which NYT influences rehabilitation training post-ICH. Material and methods 1) Animals Male Wistar rats (250–300 g) were group-housed under controlled temperature conditions (22–23°C) and a 12 h light-dark cycle, with ad libitum access to food and water. All experiments were conducted in accordance with the Animal Care guidelines of Nagoya City University Medical School and approved by the Use Committee of Nagoya City University Medical School. Every effort was made to minimize animal suffering and to reduce the number of animals used in this study. Figure 1 A illustrates the experimental procedure. In total, 268 animals were used in the experiment. Forty-seven rats were excluded because of insufficient motor dysfunction (MDS < 8 at 1 d after surgery), and 19 rats did not survive during or after surgery. 2) Model rat of ICH near the internal capsule The rat model of ICH was established according to our previous papers [ 4 , 5 , 12 , 13 , 31 , 32 ] with minor modifications. Rats were anesthetized with an intraperitoneal dose of 2 ml/kg solution of an anesthetic solution containing 0.1875 mg/ml medetomidine hydrochloride (Meiji Animal Health), 1 mg/ml midazolam hydrochloride (Fuji Pharma), and 1.25 mg/kg butorphanol tartrate (Meiji Animal Health), in conjunction with 1 ml/kg pre-treatment of 0.1 mg/kg atropine (Mitsubishi Tanabe Pharma) and 1 ml/kg post-treatment of 0.1 mg/kg dexamethasone (Aspen Japan). Following removal of the fur from the scalp, the rats were positioned in a stereotaxic frame. The left skull was exposed through a midline incision of the skin and a small hole was created using a drill in accordance with the rat atlas [ 33 ]. Type IV collagenase (1.4 µl of 15 units/ml dissolved in saline, C5138, Sigma-Aldrich) was injected in proximity to the internal capsule (1.8-mm posterior and 3.8-mm lateral to the bregma, 5-mm below the brain surface) at a rate of 0.2 µl/min over 7 min using a pulled glass capillary (tip diameter: 50–60-µm) connected to a Hamilton syringe controlled by an electric pump (ESP-64; Eicom) to minimize brain damage. After the injection, the glass capillary remained in situ for an additional 7 min to prevent backflow. The sham group underwent identical surgical procedures using an equivalent volume of saline. Subsequently, the scalp was sutured, and the rats were administered an intraperitoneal injection of 1 ml/kg solution of 0.376 mg/ml atipamezole hydrochloride (Nippon Zenyaku Kogyo) to facilitate awakening. The rodents were then allowed to recover in their home cages. One day after surgery, the extent of gross motor dysfunction was evaluated according to the motor deficit score (MDS; 0–12). Rats with an MDS of < 8 were excluded from the study to ensure a small ICH of similar hemorrhage ICH. 3) Administration of Ninjin’yoeito (NYT) Food pellets containing NYT (lot no. 39228490) were obtained from Tsumura and Co. (Tokyo, Japan). Spray-dried NYT powder was prepared from an aqueous extract of 12 medicinal herbs (Rehmannia Root, 4 g; Japanese Angelica Root, 4 g; Atractylodes Rhizome, 4 g; Poria Sclerotium, 4 g; ginseng, 3 g; Cinnamon Bark 2.5 g, Polygala Root, 2 g; Peony Root, 2 g; Citrus Unshiu Peel, 2 g; Astragalus Root 1.5 g, Glycyrrhiza 1 g; and glycyrrhiza, 1 g). Special food pellets containing NYT powder were prepared for this experiment, and the yield of the NYT powder was approximately 19%. Plant materials were authenticated through the identification of external morphology and marker compounds (glycyrrhizic acid, paeoniflorin, and hesperidin) for plant specimens, following the methods outlined in the Japanese Pharmacopeia and adhering to company standards. The quality of the extract was standardized according to good manufacturing practices as defined by the Ministry of Health, Labor, and Welfare of Japan. 4) Forced-limb Use (FLU) as a rehabilitative training Forced limb use (FLU) was employed as post-stroke rehabilitative training, as previously reported [ 12 , 13 , 31 , 32 ]. Briefly, the unimpaired forelimb and upper torso of the rats were immobilized using soft felt and plaster Paris strips under isoflurane anesthesia (2% for induction, 1% for maintenance; Mylan). Following the application of the cast, the rats were returned to their home cages and restrained for seven days, permitting minimal limb movement while preventing the use of the unimpaired limb in all daily activities. Rats in the FLU group were compelled to utilize their impaired forelimbs for all daily activities (Fig. 1 A and C). 5) Behavioral evaluation 5-1) Measurement of motor deficit score (MDS) The motor deficit score (MDS) was used to evaluate gross motor dysfunction as previously reported with minor modifications [ 4 , 31 , 34 ]. Three behavioral tests (beam walk ability, bilateral forepaw grasp, and contralateral hindlimb retraction) were conducted to assess the degree of gross motor deficit on a 5-point scale (ranging from 0 for normal to 4 for most severe dysfunction), with total scores ranging from 0 to 12. These tests were administered 1, 12, 20, 28, and 56 days after the lesion. In the beam walking test, rats were trained to traverse a wooden beam (3.0 × 3.0 × 100-cm), elevated 75-cm above the floor to return to their home cage. In the bilateral forepaw grasping test, rats were required to hold a 4-mm diameter steel rod 10 times in one trial, assessing their ability to successfully grasp the rod. In the contralateral hindlimb retraction test, the capacity to replace the hindlimb after a 20–30-mm lateral displacement was evaluated 20 times in one trial. 5-2) Horizontal ladder test The horizontal ladder apparatus comprised a 1-m long × 10-cm width ladder equipped with two transparent Plexiglas walls, which were perforated with apertures at 1-cm intervals. The ladder was positioned 75-cm above the floor and featured an unoccupied cage at the beginning and a home cage at the terminus. To evaluate the stepping function of the hindlimb, the rats were trained to traverse a 1-m-long horizontal ladder with rungs spaced regularly at 4-cm intervals to reach their home cage at a constant velocity for 3 days prior to the surgical procedure. To analyze the coordinated movement of the hindlimb on the test day, the scaffold intervals were randomly adjusted to 3-6-cm, and the rats were video-recorded while crossing the ladder. Each session consisted of three crossings, and the percentage of steps that slipped from the rungs was calculated [ 12 , 35 , 36 ]. The duration required to traverse the ladder was measured from the initial point to the terminal point over three trials. The test was performed 28 d after the lesion. 5-3) Kinematic gait analysis using pose estimation with deep learning To assess the hindlimb motor function in greater detail, gait analysis was conducted by tracking each hindlimb position using pose estimation with deep learning. The apparatus comprised a 1-m long × 10-cm width clear plexiglass flat board equipped with two clear plexiglass walls, which were positioned 75-cm above the floor and featured an empty cage at the starting point and a home cage at the terminus. The test was performed 28 d after the lesion. On the day of testing, locomotion of the rat was recorded from beneath a clear Plexiglas flat board. Each session consisted of three traversals. The angles of the hind paws in each gait were subsequently analyzed as follows: the positions of the toes and heels of the left and right paws were estimated as positional information by DeepLabCut with a deep learning technique in a markerless manner [ 37 ]. 6) Open-field test The open field test was conducted as described previously [ 38 , 39 ]. Briefly, each subject was positioned in the center of a black circular arena (60-cm diameter × 50-cm height) under standard illumination conditions (350 lx), and 30 min of unrestricted movement was recorded using a video camera mounted directly above the field. Following each test, the floor of the arena was cleaned with water to eliminate olfactory cues. A video camera was affixed directly above the open arena to record behavior for subsequent analysis. The traversed distance, frequency of entries into the central area (30-cm diameter) of the arena, and locomotion velocity were quantified using Smart software (Panlab, S.L.- Harvard Apparatus Spain) [ 40 ]. 7) Retrograde labeling of cortico-spinal neurons in the cortex To label the corticospinal tract in the sensory-motor cortex, a retrograde tracer, FluoroGold (FG; Biotium, Hayward, CA, USA), was injected into the spinal cord at the C5 level via laminectomy 28 days after ICH [ 4 ]. Under anesthesia with a mixture of medetomidine hydrochloride, midazolam, and butorphanol tartrate, 1 µL of 2% FG was administered to the center of the dorsal corticospinal tract (1-mm deep from the dura) using a pulled glass capillary (tip diameter, 50–60-µm). Following suturing of the neck, the rats were administered atipamezole hydrochloride to facilitate recovery from anesthesia. Subsequently, mice were permitted to recuperate in their cages. At D35 of 7 days after FG injection, subjects were perfused transcardially with 0.1 M PBS and 4% paraformaldehyde (PFA) under deep anesthesia with pentobarbital sodium (> 100 mg/kg, i.p.). Tokyo Chemical Industry). The brains were extracted and subjected to post-fixation and cryoprotection with 30% sucrose in 0.1 M PBS, and 40-µm-thick coronal sections from 3.2-mm anterior and 4.0-mm posterior to the bregma were subsequently prepared using a cryostat (CM 1520, Leica Microsystems). The sections were rinsed with PBS multiple times, desiccated, coverslipped with ProLong Gold Antifade Mountant (P36930, Invitrogen), and examined under a fluorescence microscope (Axioplan2, Carl Zeiss; AX70, Olympus) at 40x magnification. The total number of FG-positive cells was counted on both the ipsilateral and contralateral sides of the sensorimotor cortex using ImageJ software [ 4 ], and the data are presented as the mean ± standard error of the mean (SEM). 8) Anterograde tracer injection to detect neurites in the red nucleus Biotinylated dextran amine (BDA; MW 10,000; D1956, Thermo Fisher Scientific) was administered to the motor cortex forelimb area of the injured side as previously described [ 12 , 13 ]. Briefly, the rats were anesthetized with a combination of medetomidine hydrochloride, midazolam, and butorphanol tartrate. Subsequently, BDA (0.5µl, 5% in 0.1 M PBS) was injected at four sites (axis to bregma AP 2.5-mm, ML 2.0-mm; AP 2.5-mm, ML 3.0-mm; AP 0.5-mm, ML 2.5-mm; and AP 0.5-mm, ML 3.5-mm, each at a depth of 1.5-mm below the surface) utilizing a pulled glass capillary (tip diameter: 50–60-µm) connected to a Hamilton syringe (at a rate of 0.1 µl/min over 5 minutes) controlled by an electric pump (ESP-64; Eicom). The glass capillary was kept in position for an additional 2 min to prevent backflow. Atipamezole hydrochloride was administered to facilitate recovery from anesthesia. Animals were perfused transcardially with 0.1 M PBS and 4% PFA under deep anesthesia with pentobarbital sodium, and the brains were extracted and processed for post-fixation and cryoprotection with 30% sucrose in 0.1 M PBS. Coronal sections (40-µm-thick) were treated with 0.6% H 2 O 2 and 20% dimethyl sulfoxide (DMSO) in methanol for 30 min to inhibit endogenous peroxidase activity, and subsequently incubated with 2% ABC Elite reagent (Vector Laboratories, Burlingame, CA, USA) dissolved in PBS + 0.4% Triton X-100 (Tokyo Chemical Industry) for 2 h. After multiple washes in 10 mM Tris-buffered saline (TBS), the sections were processed in DAB-Ni reaction solution (0.01% DAB in TBS containing 1% nickel ammonium sulfate and 0.0003% H 2 O 2 ) for 30 min. Subsequently, the stained sections were counterstained with 0.5% neutral red (FUJIFILM Wako Pure Chemical Corporation), dried, and dehydrated prior to coverslipping with Entellan New (Sigma-Aldrich). The stained sections were examined and images were acquired using a light microscope (Axioplan2, Carl Zeiss; AX70, Olympus). The number of BDA-positive bouton-like varicosities in contact with neurons in the parvocellular and magnocellular regions of the red nucleus (RNp and RNm) on the lesion side was enumerated from four sections at 400x magnification, as previously described [ 12 , 13 ]. The total number of BDA-positive fibers in the cerebral peduncle was quantified from three adjacent sections rostral to the red nucleus (5.16–6.60-mm posterior to the bregma), and the counts were normalized by the uptake efficacy of BDA and lesion size. Data are presented as the mean number of bouton-like varicosities in RNp and RNm divided by the mean number of fibers counted in the cerebral peduncle for each specimen. 9) Muscle volume measurement and histochemical staining for muscle subtype To analyze the wet weight of the muscles, both gastrocnemius and soleus muscles were obtained without fixation after perfusion with 200 ml of cold PBS under deep anesthesia with pentobarbital sodium (> 100 mg/kg, i.p.). The weights were measured and expressed as the ratio of the weight of the ipsilateral side to the contralateral side in each animal. To examine morphological changes, both muscles of the gastrocnemius and the soleus that were fresh-frozen and embedded in OCT compound using liquid nitrogen were cut into 30-µm-thick cross sections, mounted on a glass slide, fixed with 4% PFA for 10 min, and processed for hematoxylin and eosin staining and immunohistochemistry for muscle type makers (MHC I, MHC IIa, MHC IIb). For hematoxylin and eosin staining, the sections were stained with Mayer's hematoxylin and eosin Y (Muto Pure Chemicals) after washing, drying, and dehydration, before being coverslipped with Entellan™ new. The stained sections were observed under a light microscope (Axioplan2, Carl Zeiss; AX70, Olympus), and random images (100x magnification) were captured for atrophy measurements. The densities of the six images from two adjacent sections were converted into binary images using the appropriate threshold values in ImageJ software. The degree of muscle atrophy is presented as the percentage of unstained area on the ipsilateral side. The average percentage in the ICH group was compared with that in each group. For immunostaining of muscle-type markers, antibodies specific to MHC I (a marker of slow-twitch), MHC IIa (a marker of mixed-twitch), and MHC IIb (a marker of fast-twitch) were utilized [ 41 – 43 ]. For the detection of slow-twitch muscles, the sections were washed with PBS containing 0.3% Triton X-100 (PBST) for 10 min in a glass container. The sections were subsequently blocked with 20% normal goat serum (NGS; Invitrogen) in PBS for 60 min at room temperature (RT) and incubated with mouse anti-MHC I IgG antibody (1:5, BA-F8, Developmental Studies Hybridoma Bank; DSHB) overnight at 4°C in a humid chamber. After five washes of 5 min each in PBS, the sections were incubated with goat anti-mouse IgG Alexa Fluor 594 (1:200; Invitrogen) for 60 min at RT. For detection of mixed-twitch muscle, mouse anti-MHC IIa IgG antibody (1:5, SC-71, DSHB) was employed as the primary antibody and goat anti-mouse IgG Alexa Fluor 405 (1:200; Invitrogen) as the secondary antibody. For the detection of fast-twitch muscle, mouse anti-MHC IIb IgM antibody (1:25, BF-F3, DSHB) served as the primary antibody, and goat anti-mouse IgM Alexa Fluor 488 (1:200; Invitrogen) as the secondary antibody. Subsequently, the sections were mounted with ProLong™ Gold Antifade Mountant (P36930, Invitrogen) and examined using a fluorescence microscope (Axioplan2, Carl Zeiss; AX70, Olympus) at 100x magnification. Representative images for each muscle subtype were obtained. The percentage area of each muscle subtype was quantified from six images captured from two adjacent sections, which were converted into binary images using appropriate threshold values in ImageJ software. The extent of each muscle subtype was expressed as the percentage of the unstained area on the ipsilateral side. The mean percentage in the ICH group was compared with that in each experimental group. The acquired images were adjusted using the remaining muscle data from hematoxylin and eosin staining. Statistics All statistical analyses were conducted using R version 4.4.0 (The R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at P < 0.05. Statistical analyses were performed to confirm disability in ICH animals using the Student's t-test. MDS changes between D1 and D28 or between D1 and D56 were evaluated using the Kruskal-Wallis rank sum test, followed by Steel's test and aligned rank transform of the factorial model (see Supplement Table 1). Other data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test for the ICH group, with the exception of the horizontal ladder test. The horizontal ladder test was performed using one-way analysis of variance (ANOVA) followed by pairwise t-tests with Bonferroni correction. Data are presented as mean ± standard error of the mean (SEM) from quintuplicates for each treatment condition. Results The effect of NYT on motor function recovery after ICH We previously reported that FLU results in functional recovery after hemorrhagic stroke in rats, demonstrating a causal relationship between the cortico-rubral pathway and functional recovery [ 12 ]. To ascertain whether NYT administration after ICH for 56 days affects hemorrhagic stroke and whether the effect of NYT is additive to the effects of FLU for 7 days (Fig. 1 A), the impact of FLU and/or NYT on gross motor functional recovery was examined by MDS in an ICH rat model exhibiting relatively severe motor dysfunction, even with small hemorrhages (Fig. 1 B and C). Five experimental groups were established: sham operation: ICH(−)-FLU(−)-NYT(−); ICH only: ICH(+)-FLU(−)-NYT(−); ICH with FLU: ICH(+)-FLU(+)-NYT(−); ICH with NYT: ICH(+)-FLU(−)-NYT(+); and ICH with both treatments: ICH(+)-FLU(+)-NYT(+). MDS exhibited a gradual decrease across all groups (Fig. 1 D). MDS at 1 d after ICH (D1) and D28 demonstrated statistically significant differences between ICH(+)-FLU(-)-NYT(-) and ICH(+)-FLU(+)-NYT(-) (p < 0.01; Fig. 1 E), indicating functional recovery induced by FLU, consistent with previous findings [ 12 ]. Functional recovery by treatment was assessed as the MDS change between D1 and D28 (Fig. 1 F) and between D1 and D56 (Fig. 1 G). While the main effect of FLU was observed at both D28 and D56, the main effect of NYT was notably demonstrated at D56 in the Kruskal-Wallis rank sum test followed by Steel's test. However, the interaction between FLU and NYT was not detected at either D28 or D56 in the aligned rank transform of the factorial model (see Supplementary Table 1), and this effect was not additive to the FLU effect. Functional recovery was shown in the hindlimb by both treatments of FLU and NYT To determine which MDS test was most effectively affected by the treatments, the change in each score was compared at D28 and D56 (Fig. 2 ). In the beam walk ability test, which is used to assess motor coordination (Fig. 2 A-C), a tendency to recover function was observed in ICH with both treatments at D28 and D56. It should be noted that the change in bilateral forelimb grasping for assessment of forelimb function was significantly increased by NYT administration at D56 in the significance was shown in ICH(+)-FLU(-)-NYT(+) and ICH (+)-FLU (+)-NYT (+) groups (Fig. 2 D-F). A tendency for better recovery was also observed in ICH with both treatments at an earlier time of D28 (p = 0.072; Fig. 2 E). In the contralateral hindlimb retraction test, which assesses hindlimb function (Fig. 2 G-I), better functional recovery (the score changes between D1 and D28 or D56) was observed in both the FLU- and NYT-treated groups compared to the ICH-only group at D28 (p < 0.01; Fig. 2 H). A tendency toward better recovery was also observed at D56 (p = 0.075; Fig. 2 I). To assess hindlimb motor function, a horizontal ladder test was also performed at D28 (Fig. 3 A-B), and the percentage of missed steps where the animal slipped from the rungs during the walk and the gate time to cross over the ladder from the starting point to the end point were analyzed (Fig. 3 C and D). Before starting the experiments, we first confirmed a significant increase in the number of missed steps in the ICH group compared with that in the sham-operated group (p < 0.001, Fig. 3 A). FLU and NYT treatments did not show significant differences in the percentage of missed steps (p = 0.220) or gait time (p = 0.970) compared to the ICH group. To achieve a more sensitive and detailed assessment of hindlimb motor function, gait analysis was performed at D28 by tracking each hindlimb position using pose estimation with deep learning (Fig. 3 E); the walking of the rat was recorded from underneath, and the angles of the hind limb paws in each gait were analyzed from the positional estimation of the toes and heels of the unimpaired left paws (α) and impaired right paws (β) using DeepLabCut. Although there is no significant difference of the balance between both hindlimbs of control non-ICH rats (the angle of β-α is 0.00 ± 0.68, n = 10), significant difference of the angle of β-α was shown in ICH-only group at D28 (6.65 ± 0.70, n = 8; p < 0.01, Fig. 3 F). Ono-way ANOVA followed by Dunnett’s multiple comparisons test revealed that FLU induced a significant decrease in the hindlimb angle of β-α (ICH-FLU(+)-NYT(-): 1.93 ± 0.68, n = 11; p < 0.05, Fig. 3 G), while NYT treatment did not induce a decrease (ICH-FLU(-)-NYT(+): 5.03 ± 0.56, n = 11, p = 0.699). The angle of β-α in both treatments (ICH-FLU(+)-NYT(+): 1.72 ± 0.56, n = 10; p < 0.05) was also significantly different from that in the ICH-only group, which was comparable to FLU treatment (Fig. 3 G). However, the interaction between FLU and NYT in hindlimb balance was not detected at D28. The effect of both treatments of FLU and NYT on emotional behavior Given that NYT has been shown to ameliorate anxiety-related behaviors and social behavior disorders in zebrafish [ 44 , 45 ], an investigation of emotional behavior was conducted using an open field test at D28 (Fig. 4 ). The total distance traveled for 30 min was 5822.95 ± 250.77 cm in the non-ICH control group (n = 9). A comparable level of total distance was observed in the ICH only group (5682.54 ± 233.24 cm, n = 8). However, treatment with both FLU and NYT resulted in a statistically significant increase in the total distance (7420.10 ± 536.79 cm, n = 8; p < 0.05) compared to the ICH-only group. Maximum speed in zone 1 was 84.87 ± 16.75 cm/s in the non-ICH sham group (n = 9), which was similar to the ICH-only group (79.38 ± 6.32 cm/s, n = 8). There was no statistically significant difference in the maximum speed in zone 1 between the groups (Fig. 4 C). In the non-ICH sham group (n = 9), the number of entries into zone 2 was significantly higher than that in the ICH-only group (6.63 ± 1.39, n = 8; p = 0.101). Although neither FLU (9.67 ± 1.80, n = 9) nor NYT (8.50 ± 1.75, n = 10) treatment increased the number of entries individually, the combined treatment group demonstrated a statistically significant increase in the number (19.50 ± 5.18, n = 8; p < 0.05) compared to the ICH-only group (Fig. 4 D). These findings suggest that the combination treatment with FLU and NYT following ICH influences emotional behavior, potentially due to enhanced spontaneous activity. The effect of both treatments of FLU and NYT on the cortical neurons in the cortico-spinal tract To analyze damage to corticospinal neurons (CSNs) in the sensorimotor cortex following ICH, FG was injected into the dorsal corticospinal tract on day 28 (D28), and labeled FG-positive cells were quantified on day 35 (D35) (Fig. 5 A). In the ICH only group, FG-positive cells were observed in the contralateral sensorimotor cortex, whereas minimal positive cells were detected in the ipsilateral cortex (ICH only, n = 6; Fig. 5 B and C), confirming that corticospinal axons were compromised in the IC model. FLU or NYT treatment did not increase the number of positive cells in the sensorimotor cortex, including in the remote area or epicenter of the injection site (FLU, n = 5; Fig. 5 D, and NYT, n = 5; Fig. 5 E). It is noteworthy that, while both FLU and NYT treatments resulted in enhanced functional recovery (Fig. 1 – 3 ) and emotional behavioral alterations (Fig. 4 ), the treatments did not elicit an increase in the number of positive cells, even in the cortex of the remote area (FLU + NYT, n = 5; Fig. 5 F). The changes of the cortico-rubral projections by the both treatments of FLU and NYT We previously reported that FLU following ICH results in functional recovery of the motor executive system accompanied by a transition from the cortico-spinal tract to the cortico-rubral tract [ 12 ]. To ascertain whether this transition was enhanced by both FLU and NYT treatments, BDA-positive varicosities, labeled by anterograde tracer BDA injection into the motor cortex forelimb area of the affected hemisphere, were quantified in both the parvocellular (RNp) and magnocellular regions (RNm) of the red nucleus (Fig. 6 ). Following FLU administration subsequent to ICH (n = 5), the ratio of BDA-positive varicosities demonstrated a statistically significant increase in both RNp (0.57 ± 0.04; p < 0.001) and RNm (0.20 ± 0.03, p < 0.01) compared to the ICH-only group (RNp: 0.15 ± 0.02, RNm; 0.07 ± 0.02) (Fig. 6 C), as previously reported [ 12 , 13 ]. The ratio in the NYT treatment group (ICH-FLU(-)-NYT(+), n = 4) remained unaltered in both RNp (0.23 ± 0.09) and RNm (0.08 ± 0.02). However, the ratio in the combined treatment group (n = 7) exhibited a statistically significant increase in RNp (0.55 ± 0.03; p < 0.001) and RNm (0.22 ± 0.03; p < 0.01), comparable to the FLU treatment group (ICH-FLU(+)-NYT(-)) (Fig. 6 C). The effect of both treatments of FLU and NYT on the muscle atrophy after ICH The weights of the gastrocnemius and soleus muscles were measured, and the ratios of ipsilateral muscle weight to contralateral muscle weight were compared among the groups (n = 4–6; Fig. 7 A-B). No statistically significant difference in the ratio was observed in the gastrocnemius (Fig. 7 A) or soleus (Fig. 7 B) muscles at D28. WWe subsequently investigated the degree of muscular atrophy using H&E staining (Fig. 7 C-D). The extent of atrophy was quantified as the percentage of the remaining muscle area within the total randomly selected area. At D28, muscle atrophy was significant in the ICH-only group (58.36 ± 4.29%; n = 6; p < 0.01) compared to the sham-operated group (88.79 ± 5.62%, n = 3) (Fig. 7 C). Although no significant difference was observed n) FLU NYT (ICH-FLU(-)-NYT(-): 64.41 ± 6.29%, n = 6), and NYT group (ICH-FLU(-)-NYT(+): 68.84 ± 8.38%, n = 6), both FLU and NYT treatments resulted in a significant increase in the percentage of remaining muscle area (84.43 ± 4.91%, n = 6; p < 0.05) (Fig. 7 D), suggesting that muscle atrophy is inhibited by both treatments at D28. We further investigated the effects of FLU and NYT on muscle subtypes using immunohistochemistry for MHC I, MHC IIa, and MHC IIb (Fig. 8 ). The percentage of the positive area for each marker in the total randomly selected area was quantified in the ICH-only group, demonstrating that the expression of MHC IIb, a marker of fast-twitch muscle, was significantly decreased after ICH (16.07 ± 8.78%, n = 6; p < 0.05) compared to sham-operation (61.7 ± 9.3%, n = 3) (Fig. 8 E). However, the expressions of a slow-twitch marker MHC I (21.64 ± 5.82%, n = 6; Fig. 8 A) and MHC IIa (30.59 ± 11.40%, n = 6; Fig. 8 C) were comparable to the sham-operated group (MHC I: 20.18 ± 2.05%, n = 3; MHC IIa: 49.74 ± 18.09%, n = 3). Notably, treatment with both FLU and NYT resulted in a statistically significant increase in the percentage of MHC IIb-positive areas after ICH at D28 (57.73 ± 6.34%, n = 6; p < 0.01), whereas FLU-only (33.88 ± 10.46%, n = 6) or NYT-only (29.24 ± 9.17%, n = 6) treatment did not elicit a significant increase after ICH (Fig. 8 F). However, no significant difference was observed in the increase of MHC I and MHC IIa expression following the combined treatments of FLU and NYT (MHC I: 33.87 ± 4.20; n = 6; MHC IIa: 50.71 ± 2.74; n = 6) compared to the ICH-only group (MHC I: 21.64 ± 5.82; n = 6; Fig. 8 B; MHC IIa: 30.59 ± 11.40; n = 6; Fig. 8 D). Discussion In addition to rehabilitative training following stroke, the significance of Kampo medicine has progressively gained recognition in stroke research [ 14 ]. Nevertheless, the efficacy of Kampo medicine NYT for hemorrhagic stroke remains uncertain, as does the potential additive effect of NYT in rehabilitative FLU training after ICH. To address these questions, we initially examined whether administration of NYT augments the effects of rehabilitative training with FLU after ICH. Subsequently, we investigated the underlying mechanism of the effect of NYT on rehabilitative training following ICH. These results indicated that long-term NYT administration following ICH led to significant functional recovery at D56, particularly in bilateral forepaw grasping. However, no synergistic effect of NYT on rehabilitative training by FLU after ICH was observed in MDS changes on both D28 and D56. The complementary effect of NYT on FLU was not evident in the gait and open field tests. Although the analysis of each MDS parameter demonstrated a tendency for the additional effect of NYT on both D28 and D56, the possibility of a synergistic effect of NYT on FLU cannot be entirely dismissed. Nevertheless, it is probable that the effect of NYT on the brain after ICH differs from that of FLU. FLU can induce a neuronal circuit switch from the cortico-spinal tract to the cortico-rubral tract, as previously reported [ 12 ]. In contrast, NYT administration alone after ICH did not alter BDA-positive varicosities in the red nucleus. Thus, the effect of NYT in ICH model rats was distinct from that of FLU. The mechanism underlying the effect of NYT on ICH appears to differ from that of FLU for several reasons. First, the switch to the corticovertebral pathway by FLU could not be enhanced by additional NYT treatment. Consequently, the ratio of BDA-positive varicosities in the NYT group was similar to that in the sham-operated group and the ratio of varicosities did not increase after FLU and NYT treatment. Secondly, the neuroprotective effect and/or anti-inflammatory effect of NYT did not appear to be substantial after ICH, although NYT containing Rehmannia Root, Japanese Angelica Root, Peony Root, and Cnidium rhizome can reduce the infarct volume in ischemic stroke rats [ 15 ]. The number of FG-positive cells in corticospinal neurons remained unchanged after NYT treatment. Third, the effect of NYT was observed in the contralateral hind limb at D28 and D56, whereas functional recovery by FLU was observed in the forelimb in our previous report [ 12 ]. NYT appears to act harmoniously on the living body, despite the complex interactions between various substances. Although the mechanism of action may be complex and challenging to elucidate, the results of our experiments should be considered empirical evidence. To date, research reports on NYT have demonstrated that NYT induces synaptic plasticity, exhibits cell-protective and anti-inflammatory effects, and affects the muscles and dopaminergic neurons. In this experiment, in addition to the FLU effect, NYT effects on synaptic plasticity, such as circuit switching from the cortico-spinal tract to the cortico-rubral tract, cell protection, and anti-inflammation were not observed. Immunohistochemical assessment of the gastrocnemius and soleus muscles for MHC I, MHC IIa, and MHC IIb indicated that supplementary NYT to FLU mitigated MHC IIb-positive fast-twitch muscle atrophy, which is characteristically observed following ICH. Therefore, it is highly plausible that the NYT effect was primarily muscular rather than neuronal, although NYT administration alone post-ICH did not elicit any alterations in MHC I, MHC IIa, or MHC IIb expression. Given that the gastrocnemius and soleus muscles possess substantial volume and play a more significant role in maintaining the animal's body than the forelimb, the observation that additional NYT treatment of FLU following ICH resulted in atrophy prevention of the fast-twitch MHC IIb fibers may be of considerable importance in the prevention of frailty. Recent studies by other research groups have reported the effect of NYT on muscle atrophy and frailty in both normal and disease model mice, demonstrating an increase in the muscle synthesis-related factors IGF-1 and IL-6 in the blood and the promotion of mTOR and 4E-BP1 phosphorylation in the soleus muscle [ 21 , 22 , 46 , 47 ]. Notably, the effect of NYT on frailty and sarcopenia has recently been documented in human subjects [ 48 – 50 ]. Therefore, it is plausible that certain factor(s) or chemical(s) present in NYT may induce trophic factors such as IGF-1 and cytokines such as IL-6 and activate mTOR and 4E-BP in fast-twitch MHC IIb fibers. Although the effect of FLU is relatively rapid, NYT requires a longer duration for its biological effect on ICH. Consequently, it would be valuable and necessary to investigate the weight of the gastrocnemius and soleus muscles, percentage area of muscle remaining, and percentage area of the changes in muscle subtypes at D56 in future studies, as these data were not observed following NYT administration at D28. As the mechanism in the brain after ICH might differ between FLU and NYT, the combination therapy of rehabilitative training by FLU with Kampo medicine by NYT potentially offers significant benefits for hemorrhagic stroke patients. Thus, short-term administration of FLU after ICH stimulates the reorganization of the remaining neural circuits, while long-term NYT administration affects fast-twitch myosin heavy chain IIb fibers in muscles, mitigating atrophy subsequent to ICH. In conclusion, this study investigated the efficacy of NYT for ICH and its potential additive effects when combined with rehabilitative training using FLU following ICH. The results clearly demonstrated that NYT affects ICH through a mechanism distinct from that of FLU, specifically by preventing atrophy of fast-twitch fibers post-ICH. Furthermore, the combination of FLU and NYT enhanced the effect of FLU on MDS changes on D28 and D56. These findings suggest the potential for combination therapy involving rehabilitative training and Kampo medicine to improve functional recovery following stroke. Declarations Competing Interests: HH was supported by a research fund from Tsumura and Co. KM was an employee of Tsumura and Co. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments This study was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (23K24714 to H.H. and 24K14226 to N.T.) and by a Grant-in-Aid for Scientific Research on Innovative Areas (“Adaptive Circuit Shift” 17H05574, and “Hyper-Adaptability” 20H05476) from the Ministry of Education, Culture, Sports, Science, and Technology (to H.H.). References Carod-Artal J, Egido JA, Gonzalez JL, Varela de Seijas E. Quality of life among stroke survivors evaluated 1 year after stroke: Experience of a stroke unit. Stroke. 2000;31:2995-3000. 10.1161/01.str.31.12.2995 Youdim MB, Ben-Shachar D, Yehuda S, Riederer P. The role of iron in the basal ganglion. Adv Neurol. 1990;53:155-62. Wagner KR, Sharp FR, Ardizzone TD, Lu A, Clark JF. Heme and iron metabolism: Role in cerebral hemorrhage. J Cereb Blood Flow Metab. 2003;23:629-52. 10.1097/01.WCB.0000073905.87928.6D Masuda T, Hida H, Kanda Y, Aihara N, Ohta K, Yamada K, et al. 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Effects of ninjin'yoeito on patients with chronic obstructive pulmonary disease and comorbid frailty and sarcopenia: A preliminary open-label randomized controlled trial. Int J Chron Obstruct Pulmon Dis. 2024;19:995-1010. 10.2147/COPD.S441767 Sakisaka N. Long-term administration of ninjin'yoeito to treat frailty in older adults: A case series. Neuropeptides. 2022;93:102244. 10.1016/j.npep.2022.102244 Arai M. Evaluating the usefulness of ninjin'yoeito kampo medicine in combination with rehabilitation therapy in patients with frailty complicated by intractable dizziness. Neuropeptides. 2021;90:102189. 10.1016/j.npep.2021.102189 Additional Declarations No competing interests reported. Supplementary Files SupplementTable1.tif Supplementary Table 1. Functional recovery was quantified by assessing changes in MDS scores between D1 and D28 as well as between D1 and D56. Statistically significant main effects of FLU were observed on both D28 and D56, whereas NYT exhibited a significant main effect on D56. No interaction effect was detected between FLU and NYT, indicating that NYT's effect is independent of FLU. Statistical analysis was conducted using the Kruskal-Wallis rank sum test with Steel's post-hoc test and aligned rank transform for factorial models. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5964788","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":413272764,"identity":"5a2cdbec-1263-457f-8652-838e29bd45f3","order_by":0,"name":"Naoki Tajiri","email":"","orcid":"","institution":"Nagoya City University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Tajiri","suffix":""},{"id":413272765,"identity":"51315df2-f6e6-483c-89a2-2cf6f44fc69a","order_by":1,"name":"Shinya Ueno","email":"","orcid":"","institution":"Nagoya City University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shinya","middleName":"","lastName":"Ueno","suffix":""},{"id":413272766,"identity":"0feff253-5df8-4b60-8436-510cd1db9b2c","order_by":2,"name":"Dewi Mustika","email":"","orcid":"","institution":"Nagoya City University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dewi","middleName":"","lastName":"Mustika","suffix":""},{"id":413272767,"identity":"0e6fa301-f8e5-4103-9b00-bc9fa2bc1193","order_by":3,"name":"Shiori Tominaga","email":"","orcid":"","institution":"Nagoya City University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shiori","middleName":"","lastName":"Tominaga","suffix":""},{"id":413272768,"identity":"c3a0bf0b-c19b-4184-9367-bdea7d959095","order_by":4,"name":"Takeshi Shimizu","email":"","orcid":"","institution":"Nagoya City University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Shimizu","suffix":""},{"id":413272769,"identity":"c17ec489-1288-47d0-85c8-f9b51ddaf06f","order_by":5,"name":"Keita Mizuno","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Keita","middleName":"","lastName":"Mizuno","suffix":""},{"id":413272771,"identity":"6847d907-fc87-4412-9661-d987c2b7fb36","order_by":6,"name":"Hideki Hida","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYBACAwnGBgmGCiDrAJoMM34tZ0jTwsAgwdiGRQtOYC7d3Hjj57xtcnwHeA9+/FJxWJ6B/YwBw48aBnZzHFos5xxstuzddttY8gBfsrTMmcOGDTw5Bow9xxiYLRtwOOxGYpsE77bbiRsO8BhIS7YdZtx/g8eAgbeBgdkAh1NBWiT/zrldD9Ri/Buoxb5BgseA8S8BLdK8DbcTDA7wmEl+bDucCNLCTMCWZmuZY7cNZx7mMbNmOJOe3MCTVnBY5pgEHr+kP7z5pua2PN/xHuObPyqsbRvYD298+KbGJhlXiCEAMOqYeRiawWygkySSDQhqAQLGHwx1cI4dUVpGwSgYBaNgJAAAS51c0+n0PVAAAAAASUVORK5CYII=","orcid":"","institution":"Nagoya City University Graduate School of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Hida","suffix":""}],"badges":[],"createdAt":"2025-02-05 10:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5964788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5964788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75890208,"identity":"0450d1f3-a1dd-4267-82e7-6d5e5d0593b8","added_by":"auto","created_at":"2025-02-10 09:44:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11609733,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of FLU and/or NYT on gross motor functional recovery in an ICH rat model.\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of experimental design. FLU was administered for 7 days from D1 to D8, and NYT was administered for 56 days from D1. Behavioral assessments were conducted using MDS on D12, D20, D28, and D56 following ICH.\u003c/p\u003e\n\u003cp\u003e(B) H\u0026amp;E staining of the ICH model showing a small hemorrhage near the internal capsule.\u003c/p\u003e\n\u003cp\u003e(C) FLU was used for post-stroke rehabilitation training. In FLU, the unimpaired forelimb and upper torso of the rats were restrained by the soft felt and plaster of Paris strips, allowing subtle limb movement but preventing the use of the unimpaired limb in all daily activities.\u003c/p\u003e\n\u003cp\u003e(D) Time course of MDS across all groups showing gradual recovery trends in ICH animals treated with both FLU and NYT: ICH(+)-FLU(+)-NYT(+).\u003c/p\u003e\n\u003cp\u003e(E) Comparison of MDS changes between D1 and D28 revealed a significant improvement with FLU (ICH(+)-FLU(+)-NYT(-)) compared to ICH-only (ICH(+)-FLU(-)-NYT(-)).\u003c/p\u003e\n\u003cp\u003e(F and G) Functional recovery by treatment was assessed as MDS change between D1 and D28 (F) and between D1 and D56 (G). The main effects of FLU (ICH(+)-FLU(+)-NYT(-)) were significant at both D28 and D56, whereas NYT exhibited a significant main effect at D56. No interaction effect between FLU and NYT was observed, suggesting that NYT was independent of FLU. Statistical analyses were performed after confirming disability in ICH animals using the Student’s t-test. Changes in MDS between D1 and D28 or D1 and D56 were evaluated using the Kruskal-Wallis rank sum test, followed by Steel’s test and aligned rank transform analysis of the factorial model. Data are presented as mean ± SEM for each treatment condition (n = 8–14). \u0026nbsp;* p \u0026lt; 0.05, ** p \u0026lt; 0.01 and **: p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/d0ba2fa3930653d9aaeb9562.png"},{"id":75886803,"identity":"d6f2f9d9-c12e-4c80-9c92-90276b2d0ece","added_by":"auto","created_at":"2025-02-10 09:20:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10773069,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of each MDS parameter showed a tendency for the additional effect of NYT at both D28 and D56.\u003c/p\u003e\n\u003cp\u003e(A-C) In the beam-walking test, a trend toward functional recovery was observed in ICH animals treated with FLU and NYT (ICH(+)-FLU(+)-NYT(+)) at both time points.\u003c/p\u003e\n\u003cp\u003e(D-F) Significant improvement in bilateral forelimb grasping induced by NYT at D56. A trend toward earlier recovery was also observed with FLU with additional NYT at D28 (p = 0.072).\u003c/p\u003e\n\u003cp\u003e(G-I) In the contralateral hindlimb retraction test, both FLU and NYT treatments significantly promoted functional recovery compared to ICH only at D28. A trend of better recovery was also observed at D56 (p = 0.075).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM for each treatment condition (n = 8–14). * p \u0026lt; 0.05 and ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/d715125e472f4adef3cb7579.png"},{"id":75886805,"identity":"e291eb74-4efc-414d-bf0a-7bcd995a444b","added_by":"auto","created_at":"2025-02-10 09:20:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12206553,"visible":true,"origin":"","legend":"\u003cp\u003eHorizontal ladder test for hindlimb motor function assessment and gait analysis, by tracking each hindlimb position using pose estimation with deep learning, was performed at D28.\u003c/p\u003e\n\u003cp\u003e(A-D) The percentage of missteps and gait time were recorded in the horizontal ladder test. Significant increases in missteps were observed in the ICH group compared to the sham group (A). However, neither FLU nor NYT significantly affected the number of steps or gait time compared with the ICH group.\u003c/p\u003e\n\u003cp\u003e(E) Using pose estimation with DeepLabCut, gait analysis was performed to assess the hindlimb motor function in more detail. The angles between the left hindlimb (non-impaired side) and the right hindlimb (impaired side) were measured as α and β, respectively.\u003c/p\u003e\n\u003cp\u003e(F) Significant differences in the β-α angle were observed in the ICH-only group at D28.\u003c/p\u003e\n\u003cp\u003e(G) FLU (ICH(+)-FLU(+)-NYT(-)) significantly decreased the hindlimb angle of β-α, whereas NYT (ICH(+)-FLU(-)-NYT(+)) showed no significant effect. FLU with additional NYT (ICH-FLU(+)-NYT(+)) showed significant improvement compared to ICH alone, but no interaction between FLU and NYT was detected at D28.\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using the Student’s t-test to confirm disability in ICH animals, followed by one-way ANOVA for the horizontal ladder test with pairwise t-tests and Bonferroni’s correction (n = 8-14). Hindlimb balance was analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test against the ICH group. Data are presented as mean ± SEM for each treatment condition (n = 8–11). * p \u0026lt; 0.05, ** p \u0026lt; 0.01 and *** p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/a5a857cb543b983a7a1b61ee.png"},{"id":75886807,"identity":"0e0ec04e-4449-4847-a812-f98ebe9cc63d","added_by":"auto","created_at":"2025-02-10 09:20:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8065768,"visible":true,"origin":"","legend":"\u003cp\u003eOpen field test for the assessment of anxiety behavior and social behavior was performed at D28.\u003c/p\u003e\n\u003cp\u003e(A) The trajectories of locomotion in each group are represented, showing the tendency of locomotion in the peripheral area (zone 1) rather than in the central area (zone2) in the unfamiliar anxious condition. (B) Both FLU and NYT treatments significantly increased the total distance traveled compared with ICH alone.\u003c/p\u003e\n\u003cp\u003e(C) The maximum speed in zone 1, a parameter for impulsivity, was similar across all groups, with no significant differences observed.\u003c/p\u003e\n\u003cp\u003e(D) The number of entries into zone 2, a parameter for anxiety, was significantly increased by additional NYT to FLU compared to ICH alone, although FLU and NYT treatment individually did not.\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using the Student’s t-test to confirm disability in ICH animals, followed by one-way ANOVA and Dunnett’s multiple comparisons test against the ICH group. Data are presented as mean ± SEM for each treatment condition (n = 8–10). * p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/24466429a623ec25b51b8927.png"},{"id":75886810,"identity":"dcb1de3b-b626-4433-83b2-ad1f93cbbd56","added_by":"auto","created_at":"2025-02-10 09:20:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11446215,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of corticospinal neuronal damage in the sensorimotor cortex following ICH.\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the experimental protocol used for assessing corticospinal neuronal damage. Fluorogold (FG) was administered into the dorsal corticospinal tract at the C5 level of the spinal cord at D28, and FG-positive cells were quantified at D35.\u003c/p\u003e\n\u003cp\u003e(B) Numerous FG-positive cell bodies are observed in the ipsilateral cortex, whereas a limited number of FG-positive cells are present in the contralateral cortex.\u003c/p\u003e\n\u003cp\u003e(C) The number of FG-positive cells in the ipsilateral and contralateral cortices is enumerated. In the ICH only group, FG-positive cells were observed in the contralateral sensorimotor cortex (white square box), whereas only a limited number of positive cells were detected in the ipsilateral cortex (black square box), thus confirming corticospinal axon damage in the ICH model. The vertical dotted line 1.8 mm dorsal from the bregma indicates the point of collagenase solution injection.\u003c/p\u003e\n\u003cp\u003e(D, E) Neither FLU nor NYT treatment increased the number of FG-positive cells in the sensorimotor cortex, encompassing both the remote and epicenter regions of the injection site.\u003c/p\u003e\n\u003cp\u003e(F) Combination treatment with FLU and NYT (FLU + NYT) also did not result in an increase in FG-positive cells, even in remote cortical areas.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM for each treatment condition (n=5-6).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/7503607f8e21fd0ba59a5e66.png"},{"id":75888707,"identity":"1c9c507a-45ca-4a89-8260-304a621ba6bb","added_by":"auto","created_at":"2025-02-10 09:36:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7468854,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of FLU and NYT treatments on BDA-positive varicosities in the red nucleus.\u003c/p\u003e\n\u003cp\u003e(A) The schematic provides an overview of the experimental design, wherein positive varicosities were labeled via anterograde tracer (BDA) injection into the motor cortex forelimb area on the injured side on D1 and subsequently quantified in the RNp and RNm regions of the red nucleus on D12.\u003c/p\u003e\n\u003cp\u003e(B) Representative images depicting BDA and neutral red staining of the cortico-rubral tract.\u003c/p\u003e\n\u003cp\u003e(C)In the FLU-treated group, the ratio of BDA-positive varicosities exhibited a statistically significant increase in both the RNp (p \u0026lt; 0.001) and RNm (p \u0026lt; 0.01) groups compared to that in the ICH-only group. The NYT treatment group (ICH-FLU(-)-NYT(+)) did not show significant alterations in either RNp or RNm. However, in the combined FLU and NYT treatment group (ICH-FLU(+)-NYT(+)), the ratio of BDA-positive varicosities was significantly elevated in both RNp (p \u0026lt; 0.001) and RNm (p \u0026lt; 0.01), reaching levels comparable to those observed with FLU treatment alone (ICH-FLU(+)-NYT(-)).\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparison test against the ICH group. Data are presented as the mean ± SEM from quintuplicate measurements for each treatment condition (n = 3–7).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/87a47d262d768a3d3890dbe9.png"},{"id":75888157,"identity":"970fe5d3-5a31-4e4a-95f3-80131b4de5bd","added_by":"auto","created_at":"2025-02-10 09:28:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9330763,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of both FLU and NYT treatments on muscle atrophy following ICH\u003c/p\u003e\n\u003cp\u003e(A, B) The weights of the gastrocnemius and soleus muscles were measured and the ratios of ipsilateral to contralateral muscle weights were compared among the groups. No statistically significant differences were observed in gastrocnemius (A) or soleus (B) muscle weight ratios at D28.\u003c/p\u003e\n\u003cp\u003e(C, D) Muscular atrophy was assessed utilizing H\u0026amp;E staining, and the extent of atrophy was quantified as the percentage of the remaining muscle area in randomly selected regions. The ICH only group exhibited significant muscle atrophy compared to the sham-operated group at D28 (C). Both FLU and NYT treatments significantly increased the percentage of the remaining muscle area, whereas no significant differences were observed in the FLU-only- and NYT-only groups (D).\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using the Student's t-test to confirm disability in ICH animals, followed by one-way ANOVA and Dunnett's multiple comparisons test against the ICH group. Data are presented as mean ± SEM for each treatment condition (n = 3–6). * P\u0026lt;0.05 and **P \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/ffa3f6fbe481c410dc7ee919.png"},{"id":75886820,"identity":"2b517c4e-99b1-4759-af03-94f82455ba8e","added_by":"auto","created_at":"2025-02-10 09:20:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":18257647,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of both FLU and NYT treatments on muscle fiber subtypes as determined by immunohistochemical analysis of MHC I, MHC IIa, and MHC IIb\u003c/p\u003e\n\u003cp\u003e(A, B) Representative images and quantification of MHC I expression demonstrating no statistically significant differences among the experimental groups.\u003c/p\u003e\n\u003cp\u003e(C, D) Representative images and quantification of MHC IIa expression showing no statistically significant differences.\u003c/p\u003e\n\u003cp\u003e(E, F) MHC IIb, a marker of fast-twitch muscle fibers, was significantly reduced in the ICH-only group compared with that in the sham-operated group (p \u0026lt; 0.05). Both FLU and NYT treatments significantly enhanced the percentage of MHC IIb-positive areas on D28 following ICH, whereas FLU- and NYT-only treatments did not result in a significant increase.\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using Student's t-test to verify disability in ICH animals, followed by one-way ANOVA and Dunnett's multiple comparisons test against the ICH group. Data are presented as the mean ± SEM from quintuplicate measurements for each treatment condition (n = 3–6).\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/0107ca7e537040e8dc672118.png"},{"id":76042656,"identity":"976de11d-4710-4460-8c09-e24d7385f388","added_by":"auto","created_at":"2025-02-11 17:32:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":76106866,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/79e6552a-a4b8-46e0-b11d-2f8618b002f0.pdf"},{"id":75886802,"identity":"c6b2a469-0aa2-4efc-a6aa-201ffdb46544","added_by":"auto","created_at":"2025-02-10 09:20:32","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1657844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e. \u0026nbsp;Functional recovery was quantified by assessing changes in MDS scores between D1 and D28 as well as between D1 and D56.\u003c/p\u003e\n\u003cp\u003eStatistically significant main effects of FLU were observed on both D28 and D56, whereas NYT exhibited a significant main effect on D56. No interaction effect was detected between FLU and NYT, indicating that NYT's effect is independent of FLU. Statistical analysis was conducted using the Kruskal-Wallis rank sum test with Steel's post-hoc test and aligned rank transform for factorial models.\u003c/p\u003e","description":"","filename":"SupplementTable1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5964788/v1/e78fd64a46887696ccb1888e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combining forced limb use with Ninjin'yoeito treatment prevents atrophy in fast-twitch muscles and promotes functional restoration after hemorrhagic stroke in rat models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHemorrhagic stroke causes impairment of motor function, leading to difficulties in daily life [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Intracerebral hemorrhage (ICH) in proximity to the internal capsule results in relatively severe motor dysfunction, even in cases of small hemorrhage [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Post-stroke rehabilitation is considered an essential strategy for mitigating motor function impairment and enhancing patients' quality of life [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Intensive utilization of paralyzed limbs, such as constraint-induced movement therapy, is available for effective rehabilitation [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The effects of rehabilitative training on the injured brain are reported to induce neurophysiological and neuroanatomical plasticity, facilitating the formation of compensatory neuronal circuits and enhanced synaptic plasticity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We have previously reported that intensive rehabilitative training of forced limb use (FLU) results in functional recovery in hemorrhagic stroke in rats, elucidating a causal link between the cortico-rubral pathway and its functional recovery [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A dynamic compensatory action to the cortico-brainstem pathways was also reported in FLU after ICH under both blockade of the corticospinal tract and the cortico-rubral tract [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to rehabilitative training after stroke, the significance of Kampo medicine has progressively been acknowledged in stroke research [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Ninjin'yoeito (NYT) is a traditional Japanese Kampo medicine that affects the brain and muscle. Notably, the component crude-drugs of NYT, such as Rehmannia Root, Japanese Angelica Root, Peony Root, and Cnidium rhizome, have been demonstrated to reduce the infarct volume in ischemic stroke rats [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. From a clinical perspective, NYT offers various benefits, including alleviation of fatigue, malaise, anorexia, frailty, sarcopenia, and cognitive dysfunction [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, NYT enhances motivation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], augments dopamine signaling [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], promotes remyelination [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and facilitates the synthesis and secretion of nerve growth factor (NGF) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although interest in Kampo medicine for rehabilitative training is increasing [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], it remains unclear whether NYT itself has an effect on hemorrhagic stroke and whether NYT has an additive effect on rehabilitative training after ICH.\u003c/p\u003e \u003cp\u003eIn this investigation, we initially ascertained whether the administration of NYT augments the effects of rehabilitative training by FLU following hemorrhagic stroke, using an ICH rat model exhibiting relatively severe motor dysfunction even with small hemorrhages. Subsequently, we examined the underlying mechanism by which NYT influences rehabilitation training post-ICH.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1) Animals\u003c/h2\u003e \u003cp\u003eMale Wistar rats (250\u0026ndash;300 g) were group-housed under controlled temperature conditions (22\u0026ndash;23\u0026deg;C) and a 12 h light-dark cycle, with ad libitum access to food and water. All experiments were conducted in accordance with the Animal Care guidelines of Nagoya City University Medical School and approved by the Use Committee of Nagoya City University Medical School. Every effort was made to minimize animal suffering and to reduce the number of animals used in this study. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA illustrates the experimental procedure. In total, 268 animals were used in the experiment. Forty-seven rats were excluded because of insufficient motor dysfunction (MDS\u0026thinsp;\u0026lt;\u0026thinsp;8 at 1 d after surgery), and 19 rats did not survive during or after surgery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2) Model rat of ICH near the internal capsule\u003c/h3\u003e\n\u003cp\u003eThe rat model of ICH was established according to our previous papers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] with minor modifications. Rats were anesthetized with an intraperitoneal dose of 2 ml/kg solution of an anesthetic solution containing 0.1875 mg/ml medetomidine hydrochloride (Meiji Animal Health), 1 mg/ml midazolam hydrochloride (Fuji Pharma), and 1.25 mg/kg butorphanol tartrate (Meiji Animal Health), in conjunction with 1 ml/kg pre-treatment of 0.1 mg/kg atropine (Mitsubishi Tanabe Pharma) and 1 ml/kg post-treatment of 0.1 mg/kg dexamethasone (Aspen Japan).\u003c/p\u003e \u003cp\u003eFollowing removal of the fur from the scalp, the rats were positioned in a stereotaxic frame. The left skull was exposed through a midline incision of the skin and a small hole was created using a drill in accordance with the rat atlas [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Type IV collagenase (1.4 \u0026micro;l of 15 units/ml dissolved in saline, C5138, Sigma-Aldrich) was injected in proximity to the internal capsule (1.8-mm posterior and 3.8-mm lateral to the bregma, 5-mm below the brain surface) at a rate of 0.2 \u0026micro;l/min over 7 min using a pulled glass capillary (tip diameter: 50\u0026ndash;60-\u0026micro;m) connected to a Hamilton syringe controlled by an electric pump (ESP-64; Eicom) to minimize brain damage. After the injection, the glass capillary remained in situ for an additional 7 min to prevent backflow. The sham group underwent identical surgical procedures using an equivalent volume of saline. Subsequently, the scalp was sutured, and the rats were administered an intraperitoneal injection of 1 ml/kg solution of 0.376 mg/ml atipamezole hydrochloride (Nippon Zenyaku Kogyo) to facilitate awakening. The rodents were then allowed to recover in their home cages. One day after surgery, the extent of gross motor dysfunction was evaluated according to the motor deficit score (MDS; 0\u0026ndash;12). Rats with an MDS of \u0026lt;\u0026thinsp;8 were excluded from the study to ensure a small ICH of similar hemorrhage ICH.\u003c/p\u003e\n\u003ch3\u003e3) Administration of Ninjin’yoeito (NYT)\u003c/h3\u003e\n\u003cp\u003eFood pellets containing NYT (lot no. 39228490) were obtained from Tsumura and Co. (Tokyo, Japan). Spray-dried NYT powder was prepared from an aqueous extract of 12 medicinal herbs (Rehmannia Root, 4 g; Japanese Angelica Root, 4 g; Atractylodes Rhizome, 4 g; Poria Sclerotium, 4 g; ginseng, 3 g; Cinnamon Bark 2.5 g, Polygala Root, 2 g; Peony Root, 2 g; Citrus Unshiu Peel, 2 g; Astragalus Root 1.5 g, Glycyrrhiza 1 g; and glycyrrhiza, 1 g). Special food pellets containing NYT powder were prepared for this experiment, and the yield of the NYT powder was approximately 19%. Plant materials were authenticated through the identification of external morphology and marker compounds (glycyrrhizic acid, paeoniflorin, and hesperidin) for plant specimens, following the methods outlined in the Japanese Pharmacopeia and adhering to company standards. The quality of the extract was standardized according to good manufacturing practices as defined by the Ministry of Health, Labor, and Welfare of Japan.\u003c/p\u003e\n\u003ch3\u003e4) Forced-limb Use (FLU) as a rehabilitative training\u003c/h3\u003e\n\u003cp\u003eForced limb use (FLU) was employed as post-stroke rehabilitative training, as previously reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Briefly, the unimpaired forelimb and upper torso of the rats were immobilized using soft felt and plaster Paris strips under isoflurane anesthesia (2% for induction, 1% for maintenance; Mylan). Following the application of the cast, the rats were returned to their home cages and restrained for seven days, permitting minimal limb movement while preventing the use of the unimpaired limb in all daily activities. Rats in the FLU group were compelled to utilize their impaired forelimbs for all daily activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and C).\u003c/p\u003e\n\u003ch3\u003e5) Behavioral evaluation\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e5-1) Measurement of motor deficit score (MDS)\u003c/h2\u003e \u003cp\u003eThe motor deficit score (MDS) was used to evaluate gross motor dysfunction as previously reported with minor modifications [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Three behavioral tests (beam walk ability, bilateral forepaw grasp, and contralateral hindlimb retraction) were conducted to assess the degree of gross motor deficit on a 5-point scale (ranging from 0 for normal to 4 for most severe dysfunction), with total scores ranging from 0 to 12. These tests were administered 1, 12, 20, 28, and 56 days after the lesion. In the beam walking test, rats were trained to traverse a wooden beam (3.0 \u0026times; 3.0 \u0026times; 100-cm), elevated 75-cm above the floor to return to their home cage. In the bilateral forepaw grasping test, rats were required to hold a 4-mm diameter steel rod 10 times in one trial, assessing their ability to successfully grasp the rod. In the contralateral hindlimb retraction test, the capacity to replace the hindlimb after a 20\u0026ndash;30-mm lateral displacement was evaluated 20 times in one trial.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e5-2) Horizontal ladder test\u003c/h3\u003e\n\u003cp\u003eThe horizontal ladder apparatus comprised a 1-m long \u0026times; 10-cm width ladder equipped with two transparent Plexiglas walls, which were perforated with apertures at 1-cm intervals. The ladder was positioned 75-cm above the floor and featured an unoccupied cage at the beginning and a home cage at the terminus. To evaluate the stepping function of the hindlimb, the rats were trained to traverse a 1-m-long horizontal ladder with rungs spaced regularly at 4-cm intervals to reach their home cage at a constant velocity for 3 days prior to the surgical procedure. To analyze the coordinated movement of the hindlimb on the test day, the scaffold intervals were randomly adjusted to 3-6-cm, and the rats were video-recorded while crossing the ladder. Each session consisted of three crossings, and the percentage of steps that slipped from the rungs was calculated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The duration required to traverse the ladder was measured from the initial point to the terminal point over three trials. The test was performed 28 d after the lesion.\u003c/p\u003e\n\u003ch3\u003e5-3) Kinematic gait analysis using pose estimation with deep learning\u003c/h3\u003e\n\u003cp\u003eTo assess the hindlimb motor function in greater detail, gait analysis was conducted by tracking each hindlimb position using pose estimation with deep learning. The apparatus comprised a 1-m long \u0026times; 10-cm width clear plexiglass flat board equipped with two clear plexiglass walls, which were positioned 75-cm above the floor and featured an empty cage at the starting point and a home cage at the terminus. The test was performed 28 d after the lesion. On the day of testing, locomotion of the rat was recorded from beneath a clear Plexiglas flat board. Each session consisted of three traversals. The angles of the hind paws in each gait were subsequently analyzed as follows: the positions of the toes and heels of the left and right paws were estimated as positional information by DeepLabCut with a deep learning technique in a markerless manner [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e6) Open-field test\u003c/h2\u003e \u003cp\u003eThe open field test was conducted as described previously [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Briefly, each subject was positioned in the center of a black circular arena (60-cm diameter \u0026times; 50-cm height) under standard illumination conditions (350 lx), and 30 min of unrestricted movement was recorded using a video camera mounted directly above the field. Following each test, the floor of the arena was cleaned with water to eliminate olfactory cues. A video camera was affixed directly above the open arena to record behavior for subsequent analysis. The traversed distance, frequency of entries into the central area (30-cm diameter) of the arena, and locomotion velocity were quantified using Smart software (Panlab, S.L.- Harvard Apparatus Spain) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e7) Retrograde labeling of cortico-spinal neurons in the cortex\u003c/h2\u003e \u003cp\u003eTo label the corticospinal tract in the sensory-motor cortex, a retrograde tracer, FluoroGold (FG; Biotium, Hayward, CA, USA), was injected into the spinal cord at the C5 level via laminectomy 28 days after ICH [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Under anesthesia with a mixture of medetomidine hydrochloride, midazolam, and butorphanol tartrate, 1 \u0026micro;L of 2% FG was administered to the center of the dorsal corticospinal tract (1-mm deep from the dura) using a pulled glass capillary (tip diameter, 50\u0026ndash;60-\u0026micro;m). Following suturing of the neck, the rats were administered atipamezole hydrochloride to facilitate recovery from anesthesia. Subsequently, mice were permitted to recuperate in their cages.\u003c/p\u003e \u003cp\u003eAt D35 of 7 days after FG injection, subjects were perfused transcardially with 0.1 M PBS and 4% paraformaldehyde (PFA) under deep anesthesia with pentobarbital sodium (\u0026gt;\u0026thinsp;100 mg/kg, i.p.). Tokyo Chemical Industry). The brains were extracted and subjected to post-fixation and cryoprotection with 30% sucrose in 0.1 M PBS, and 40-\u0026micro;m-thick coronal sections from 3.2-mm anterior and 4.0-mm posterior to the bregma were subsequently prepared using a cryostat (CM 1520, Leica Microsystems). The sections were rinsed with PBS multiple times, desiccated, coverslipped with ProLong Gold Antifade Mountant (P36930, Invitrogen), and examined under a fluorescence microscope (Axioplan2, Carl Zeiss; AX70, Olympus) at 40x magnification.\u003c/p\u003e \u003cp\u003eThe total number of FG-positive cells was counted on both the ipsilateral and contralateral sides of the sensorimotor cortex using ImageJ software [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and the data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e8) Anterograde tracer injection to detect neurites in the red nucleus\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBiotinylated dextran amine (BDA; MW 10,000; D1956, Thermo Fisher Scientific) was administered to the motor cortex forelimb area of the injured side as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, the rats were anesthetized with a combination of medetomidine hydrochloride, midazolam, and butorphanol tartrate. Subsequently, BDA (0.5\u0026micro;l, 5% in 0.1 M PBS) was injected at four sites (axis to bregma AP 2.5-mm, ML 2.0-mm; AP 2.5-mm, ML 3.0-mm; AP 0.5-mm, ML 2.5-mm; and AP 0.5-mm, ML 3.5-mm, each at a depth of 1.5-mm below the surface) utilizing a pulled glass capillary (tip diameter: 50\u0026ndash;60-\u0026micro;m) connected to a Hamilton syringe (at a rate of 0.1 \u0026micro;l/min over 5 minutes) controlled by an electric pump (ESP-64; Eicom). The glass capillary was kept in position for an additional 2 min to prevent backflow. Atipamezole hydrochloride was administered to facilitate recovery from anesthesia.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnimals were perfused transcardially with 0.1 M PBS and 4% PFA under deep anesthesia with pentobarbital sodium, and the brains were extracted and processed for post-fixation and cryoprotection with 30% sucrose in 0.1 M PBS. Coronal sections (40-\u0026micro;m-thick) were treated with 0.6% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 20% dimethyl sulfoxide (DMSO) in methanol for 30 min to inhibit endogenous peroxidase activity, and subsequently incubated with 2% ABC Elite reagent (Vector Laboratories, Burlingame, CA, USA) dissolved in PBS\u0026thinsp;+\u0026thinsp;0.4% Triton X-100 (Tokyo Chemical Industry) for 2 h. After multiple washes in 10 mM Tris-buffered saline (TBS), the sections were processed in DAB-Ni reaction solution (0.01% DAB in TBS containing 1% nickel ammonium sulfate and 0.0003% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) for 30 min. Subsequently, the stained sections were counterstained with 0.5% neutral red (FUJIFILM Wako Pure Chemical Corporation), dried, and dehydrated prior to coverslipping with Entellan New (Sigma-Aldrich). The stained sections were examined and images were acquired using a light microscope (Axioplan2, Carl Zeiss; AX70, Olympus).\u003c/p\u003e \u003cp\u003eThe number of BDA-positive bouton-like varicosities in contact with neurons in the parvocellular and magnocellular regions of the red nucleus (RNp and RNm) on the lesion side was enumerated from four sections at 400x magnification, as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The total number of BDA-positive fibers in the cerebral peduncle was quantified from three adjacent sections rostral to the red nucleus (5.16\u0026ndash;6.60-mm posterior to the bregma), and the counts were normalized by the uptake efficacy of BDA and lesion size. Data are presented as the mean number of bouton-like varicosities in RNp and RNm divided by the mean number of fibers counted in the cerebral peduncle for each specimen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e9) Muscle volume measurement and histochemical staining for muscle subtype\u003c/h2\u003e \u003cp\u003eTo analyze the wet weight of the muscles, both gastrocnemius and soleus muscles were obtained without fixation after perfusion with 200 ml of cold PBS under deep anesthesia with pentobarbital sodium (\u0026gt;\u0026thinsp;100 mg/kg, i.p.). The weights were measured and expressed as the ratio of the weight of the ipsilateral side to the contralateral side in each animal.\u003c/p\u003e \u003cp\u003eTo examine morphological changes, both muscles of the gastrocnemius and the soleus that were fresh-frozen and embedded in OCT compound using liquid nitrogen were cut into 30-\u0026micro;m-thick cross sections, mounted on a glass slide, fixed with 4% PFA for 10 min, and processed for hematoxylin and eosin staining and immunohistochemistry for muscle type makers (MHC I, MHC IIa, MHC IIb). For hematoxylin and eosin staining, the sections were stained with Mayer's hematoxylin and eosin Y (Muto Pure Chemicals) after washing, drying, and dehydration, before being coverslipped with Entellan\u0026trade; new. The stained sections were observed under a light microscope (Axioplan2, Carl Zeiss; AX70, Olympus), and random images (100x magnification) were captured for atrophy measurements. The densities of the six images from two adjacent sections were converted into binary images using the appropriate threshold values in ImageJ software. The degree of muscle atrophy is presented as the percentage of unstained area on the ipsilateral side. The average percentage in the ICH group was compared with that in each group.\u003c/p\u003e \u003cp\u003eFor immunostaining of muscle-type markers, antibodies specific to MHC I (a marker of slow-twitch), MHC IIa (a marker of mixed-twitch), and MHC IIb (a marker of fast-twitch) were utilized [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. For the detection of slow-twitch muscles, the sections were washed with PBS containing 0.3% Triton X-100 (PBST) for 10 min in a glass container. The sections were subsequently blocked with 20% normal goat serum (NGS; Invitrogen) in PBS for 60 min at room temperature (RT) and incubated with mouse anti-MHC I IgG antibody (1:5, BA-F8, Developmental Studies Hybridoma Bank; DSHB) overnight at 4\u0026deg;C in a humid chamber. After five washes of 5 min each in PBS, the sections were incubated with goat anti-mouse IgG Alexa Fluor 594 (1:200; Invitrogen) for 60 min at RT. For detection of mixed-twitch muscle, mouse anti-MHC IIa IgG antibody (1:5, SC-71, DSHB) was employed as the primary antibody and goat anti-mouse IgG Alexa Fluor 405 (1:200; Invitrogen) as the secondary antibody. For the detection of fast-twitch muscle, mouse anti-MHC IIb IgM antibody (1:25, BF-F3, DSHB) served as the primary antibody, and goat anti-mouse IgM Alexa Fluor 488 (1:200; Invitrogen) as the secondary antibody.\u003c/p\u003e \u003cp\u003eSubsequently, the sections were mounted with ProLong\u0026trade; Gold Antifade Mountant (P36930, Invitrogen) and examined using a fluorescence microscope (Axioplan2, Carl Zeiss; AX70, Olympus) at 100x magnification. Representative images for each muscle subtype were obtained. The percentage area of each muscle subtype was quantified from six images captured from two adjacent sections, which were converted into binary images using appropriate threshold values in ImageJ software. The extent of each muscle subtype was expressed as the percentage of the unstained area on the ipsilateral side. The mean percentage in the ICH group was compared with that in each experimental group. The acquired images were adjusted using the remaining muscle data from hematoxylin and eosin staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using R version 4.4.0 (The R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eStatistical analyses were performed to confirm disability in ICH animals using the Student's t-test. MDS changes between D1 and D28 or between D1 and D56 were evaluated using the Kruskal-Wallis rank sum test, followed by Steel's test and aligned rank transform of the factorial model (see Supplement Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eOther data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test for the ICH group, with the exception of the horizontal ladder test. The horizontal ladder test was performed using one-way analysis of variance (ANOVA) followed by pairwise t-tests with Bonferroni correction. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) from quintuplicates for each treatment condition.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of NYT on motor function recovery after ICH\u003c/h2\u003e \u003cp\u003eWe previously reported that FLU results in functional recovery after hemorrhagic stroke in rats, demonstrating a causal relationship between the cortico-rubral pathway and functional recovery [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To ascertain whether NYT administration after ICH for 56 days affects hemorrhagic stroke and whether the effect of NYT is additive to the effects of FLU for 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), the impact of FLU and/or NYT on gross motor functional recovery was examined by MDS in an ICH rat model exhibiting relatively severe motor dysfunction, even with small hemorrhages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003eFive experimental groups were established: sham operation: ICH(\u0026minus;)-FLU(\u0026minus;)-NYT(\u0026minus;); ICH only: ICH(+)-FLU(\u0026minus;)-NYT(\u0026minus;); ICH with FLU: ICH(+)-FLU(+)-NYT(\u0026minus;); ICH with NYT: ICH(+)-FLU(\u0026minus;)-NYT(+); and ICH with both treatments: ICH(+)-FLU(+)-NYT(+). MDS exhibited a gradual decrease across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). MDS at 1 d after ICH (D1) and D28 demonstrated statistically significant differences between ICH(+)-FLU(-)-NYT(-) and ICH(+)-FLU(+)-NYT(-) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), indicating functional recovery induced by FLU, consistent with previous findings [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFunctional recovery by treatment was assessed as the MDS change between D1 and D28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and between D1 and D56 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). While the main effect of FLU was observed at both D28 and D56, the main effect of NYT was notably demonstrated at D56 in the Kruskal-Wallis rank sum test followed by Steel's test. However, the interaction between FLU and NYT was not detected at either D28 or D56 in the aligned rank transform of the factorial model (see Supplementary Table\u0026nbsp;1), and this effect was not additive to the FLU effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFunctional recovery was shown in the hindlimb by both treatments of FLU and NYT\u003c/h2\u003e \u003cp\u003eTo determine which MDS test was most effectively affected by the treatments, the change in each score was compared at D28 and D56 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the beam walk ability test, which is used to assess motor coordination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C), a tendency to recover function was observed in ICH with both treatments at D28 and D56. It should be noted that the change in bilateral forelimb grasping for assessment of forelimb function was significantly increased by NYT administration at D56 in the significance was shown in ICH(+)-FLU(-)-NYT(+) and ICH (+)-FLU (+)-NYT (+) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). A tendency for better recovery was also observed in ICH with both treatments at an earlier time of D28 (p\u0026thinsp;=\u0026thinsp;0.072; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In the contralateral hindlimb retraction test, which assesses hindlimb function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-I), better functional recovery (the score changes between D1 and D28 or D56) was observed in both the FLU- and NYT-treated groups compared to the ICH-only group at D28 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). A tendency toward better recovery was also observed at D56 (p\u0026thinsp;=\u0026thinsp;0.075; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eTo assess hindlimb motor function, a horizontal ladder test was also performed at D28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B), and the percentage of missed steps where the animal slipped from the rungs during the walk and the gate time to cross over the ladder from the starting point to the end point were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). Before starting the experiments, we first confirmed a significant increase in the number of missed steps in the ICH group compared with that in the sham-operated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). FLU and NYT treatments did not show significant differences in the percentage of missed steps (p\u0026thinsp;=\u0026thinsp;0.220) or gait time (p\u0026thinsp;=\u0026thinsp;0.970) compared to the ICH group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo achieve a more sensitive and detailed assessment of hindlimb motor function, gait analysis was performed at D28 by tracking each hindlimb position using pose estimation with deep learning (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE); the walking of the rat was recorded from underneath, and the angles of the hind limb paws in each gait were analyzed from the positional estimation of the toes and heels of the unimpaired left paws (α) and impaired right paws (β) using DeepLabCut.\u003c/p\u003e \u003cp\u003eAlthough there is no significant difference of the balance between both hindlimbs of control non-ICH rats (the angle of β-α is 0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68, n\u0026thinsp;=\u0026thinsp;10), significant difference of the angle of β-α was shown in ICH-only group at D28 (6.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70, n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Ono-way ANOVA followed by Dunnett\u0026rsquo;s multiple comparisons test revealed that FLU induced a significant decrease in the hindlimb angle of β-α (ICH-FLU(+)-NYT(-): 1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68, n\u0026thinsp;=\u0026thinsp;11; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), while NYT treatment did not induce a decrease (ICH-FLU(-)-NYT(+): 5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56, n\u0026thinsp;=\u0026thinsp;11, p\u0026thinsp;=\u0026thinsp;0.699). The angle of β-α in both treatments (ICH-FLU(+)-NYT(+): 1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56, n\u0026thinsp;=\u0026thinsp;10; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was also significantly different from that in the ICH-only group, which was comparable to FLU treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). However, the interaction between FLU and NYT in hindlimb balance was not detected at D28.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of both treatments of FLU and NYT on emotional behavior\u003c/h2\u003e \u003cp\u003eGiven that NYT has been shown to ameliorate anxiety-related behaviors and social behavior disorders in zebrafish [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], an investigation of emotional behavior was conducted using an open field test at D28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe total distance traveled for 30 min was 5822.95\u0026thinsp;\u0026plusmn;\u0026thinsp;250.77 cm in the non-ICH control group (n\u0026thinsp;=\u0026thinsp;9). A comparable level of total distance was observed in the ICH only group (5682.54\u0026thinsp;\u0026plusmn;\u0026thinsp;233.24 cm, n\u0026thinsp;=\u0026thinsp;8). However, treatment with both FLU and NYT resulted in a statistically significant increase in the total distance (7420.10\u0026thinsp;\u0026plusmn;\u0026thinsp;536.79 cm, n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the ICH-only group. Maximum speed in zone 1 was 84.87\u0026thinsp;\u0026plusmn;\u0026thinsp;16.75 cm/s in the non-ICH sham group (n\u0026thinsp;=\u0026thinsp;9), which was similar to the ICH-only group (79.38\u0026thinsp;\u0026plusmn;\u0026thinsp;6.32 cm/s, n\u0026thinsp;=\u0026thinsp;8). There was no statistically significant difference in the maximum speed in zone 1 between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In the non-ICH sham group (n\u0026thinsp;=\u0026thinsp;9), the number of entries into zone 2 was significantly higher than that in the ICH-only group (6.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39, n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;=\u0026thinsp;0.101). Although neither FLU (9.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80, n\u0026thinsp;=\u0026thinsp;9) nor NYT (8.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75, n\u0026thinsp;=\u0026thinsp;10) treatment increased the number of entries individually, the combined treatment group demonstrated a statistically significant increase in the number (19.50\u0026thinsp;\u0026plusmn;\u0026thinsp;5.18, n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the ICH-only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThese findings suggest that the combination treatment with FLU and NYT following ICH influences emotional behavior, potentially due to enhanced spontaneous activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effect of both treatments of FLU and NYT on the cortical neurons in the cortico-spinal tract\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo analyze damage to corticospinal neurons (CSNs) in the sensorimotor cortex following ICH, FG was injected into the dorsal corticospinal tract on day 28 (D28), and labeled FG-positive cells were quantified on day 35 (D35) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the ICH only group, FG-positive cells were observed in the contralateral sensorimotor cortex, whereas minimal positive cells were detected in the ipsilateral cortex (ICH only, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C), confirming that corticospinal axons were compromised in the IC model. FLU or NYT treatment did not increase the number of positive cells in the sensorimotor cortex, including in the remote area or epicenter of the injection site (FLU, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, and NYT, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). It is noteworthy that, while both FLU and NYT treatments resulted in enhanced functional recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and emotional behavioral alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the treatments did not elicit an increase in the number of positive cells, even in the cortex of the remote area (FLU\u0026thinsp;+\u0026thinsp;NYT, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe changes of the cortico-rubral projections by the both treatments of FLU and NYT\u003c/h2\u003e \u003cp\u003eWe previously reported that FLU following ICH results in functional recovery of the motor executive system accompanied by a transition from the cortico-spinal tract to the cortico-rubral tract [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To ascertain whether this transition was enhanced by both FLU and NYT treatments, BDA-positive varicosities, labeled by anterograde tracer BDA injection into the motor cortex forelimb area of the affected hemisphere, were quantified in both the parvocellular (RNp) and magnocellular regions (RNm) of the red nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing FLU administration subsequent to ICH (n\u0026thinsp;=\u0026thinsp;5), the ratio of BDA-positive varicosities demonstrated a statistically significant increase in both RNp (0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and RNm (0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the ICH-only group (RNp: 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, RNm; 0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), as previously reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The ratio in the NYT treatment group (ICH-FLU(-)-NYT(+), n\u0026thinsp;=\u0026thinsp;4) remained unaltered in both RNp (0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09) and RNm (0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02). However, the ratio in the combined treatment group (n\u0026thinsp;=\u0026thinsp;7) exhibited a statistically significant increase in RNp (0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and RNm (0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), comparable to the FLU treatment group (ICH-FLU(+)-NYT(-)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of both treatments of FLU and NYT on the muscle atrophy after ICH\u003c/h2\u003e \u003cp\u003eThe weights of the gastrocnemius and soleus muscles were measured, and the ratios of ipsilateral muscle weight to contralateral muscle weight were compared among the groups (n\u0026thinsp;=\u0026thinsp;4\u0026ndash;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). No statistically significant difference in the ratio was observed in the gastrocnemius (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) or soleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) muscles at D28.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWWe subsequently investigated the degree of muscular atrophy using H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). The extent of atrophy was quantified as the percentage of the remaining muscle area within the total randomly selected area. At D28, muscle atrophy was significant in the ICH-only group (58.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.29%; n\u0026thinsp;=\u0026thinsp;6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the sham-operated group (88.79\u0026thinsp;\u0026plusmn;\u0026thinsp;5.62%, n\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Although no significant difference was observed n) FLU NYT (ICH-FLU(-)-NYT(-): 64.41\u0026thinsp;\u0026plusmn;\u0026thinsp;6.29%, n\u0026thinsp;=\u0026thinsp;6), and NYT group (ICH-FLU(-)-NYT(+): 68.84\u0026thinsp;\u0026plusmn;\u0026thinsp;8.38%, n\u0026thinsp;=\u0026thinsp;6), both FLU and NYT treatments resulted in a significant increase in the percentage of remaining muscle area (84.43\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91%, n\u0026thinsp;=\u0026thinsp;6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), suggesting that muscle atrophy is inhibited by both treatments at D28.\u003c/p\u003e \u003cp\u003eWe further investigated the effects of FLU and NYT on muscle subtypes using immunohistochemistry for MHC I, MHC IIa, and MHC IIb (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The percentage of the positive area for each marker in the total randomly selected area was quantified in the ICH-only group, demonstrating that the expression of MHC IIb, a marker of fast-twitch muscle, was significantly decreased after ICH (16.07\u0026thinsp;\u0026plusmn;\u0026thinsp;8.78%, n\u0026thinsp;=\u0026thinsp;6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to sham-operation (61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3%, n\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). However, the expressions of a slow-twitch marker MHC I (21.64\u0026thinsp;\u0026plusmn;\u0026thinsp;5.82%, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) and MHC IIa (30.59\u0026thinsp;\u0026plusmn;\u0026thinsp;11.40%, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) were comparable to the sham-operated group (MHC I: 20.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05%, n\u0026thinsp;=\u0026thinsp;3; MHC IIa: 49.74\u0026thinsp;\u0026plusmn;\u0026thinsp;18.09%, n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, treatment with both FLU and NYT resulted in a statistically significant increase in the percentage of MHC IIb-positive areas after ICH at D28 (57.73\u0026thinsp;\u0026plusmn;\u0026thinsp;6.34%, n\u0026thinsp;=\u0026thinsp;6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas FLU-only (33.88\u0026thinsp;\u0026plusmn;\u0026thinsp;10.46%, n\u0026thinsp;=\u0026thinsp;6) or NYT-only (29.24\u0026thinsp;\u0026plusmn;\u0026thinsp;9.17%, n\u0026thinsp;=\u0026thinsp;6) treatment did not elicit a significant increase after ICH (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). However, no significant difference was observed in the increase of MHC I and MHC IIa expression following the combined treatments of FLU and NYT (MHC I: 33.87\u0026thinsp;\u0026plusmn;\u0026thinsp;4.20; n\u0026thinsp;=\u0026thinsp;6; MHC IIa: 50.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74; n\u0026thinsp;=\u0026thinsp;6) compared to the ICH-only group (MHC I: 21.64\u0026thinsp;\u0026plusmn;\u0026thinsp;5.82; n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB; MHC IIa: 30.59\u0026thinsp;\u0026plusmn;\u0026thinsp;11.40; n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn addition to rehabilitative training following stroke, the significance of Kampo medicine has progressively gained recognition in stroke research [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nevertheless, the efficacy of Kampo medicine NYT for hemorrhagic stroke remains uncertain, as does the potential additive effect of NYT in rehabilitative FLU training after ICH. To address these questions, we initially examined whether administration of NYT augments the effects of rehabilitative training with FLU after ICH. Subsequently, we investigated the underlying mechanism of the effect of NYT on rehabilitative training following ICH.\u003c/p\u003e \u003cp\u003eThese results indicated that long-term NYT administration following ICH led to significant functional recovery at D56, particularly in bilateral forepaw grasping. However, no synergistic effect of NYT on rehabilitative training by FLU after ICH was observed in MDS changes on both D28 and D56. The complementary effect of NYT on FLU was not evident in the gait and open field tests. Although the analysis of each MDS parameter demonstrated a tendency for the additional effect of NYT on both D28 and D56, the possibility of a synergistic effect of NYT on FLU cannot be entirely dismissed. Nevertheless, it is probable that the effect of NYT on the brain after ICH differs from that of FLU. FLU can induce a neuronal circuit switch from the cortico-spinal tract to the cortico-rubral tract, as previously reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, NYT administration alone after ICH did not alter BDA-positive varicosities in the red nucleus. Thus, the effect of NYT in ICH model rats was distinct from that of FLU.\u003c/p\u003e \u003cp\u003eThe mechanism underlying the effect of NYT on ICH appears to differ from that of FLU for several reasons. First, the switch to the corticovertebral pathway by FLU could not be enhanced by additional NYT treatment. Consequently, the ratio of BDA-positive varicosities in the NYT group was similar to that in the sham-operated group and the ratio of varicosities did not increase after FLU and NYT treatment. Secondly, the neuroprotective effect and/or anti-inflammatory effect of NYT did not appear to be substantial after ICH, although NYT containing Rehmannia Root, Japanese Angelica Root, Peony Root, and Cnidium rhizome can reduce the infarct volume in ischemic stroke rats [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The number of FG-positive cells in corticospinal neurons remained unchanged after NYT treatment. Third, the effect of NYT was observed in the contralateral hind limb at D28 and D56, whereas functional recovery by FLU was observed in the forelimb in our previous report [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. NYT appears to act harmoniously on the living body, despite the complex interactions between various substances. Although the mechanism of action may be complex and challenging to elucidate, the results of our experiments should be considered empirical evidence. To date, research reports on NYT have demonstrated that NYT induces synaptic plasticity, exhibits cell-protective and anti-inflammatory effects, and affects the muscles and dopaminergic neurons. In this experiment, in addition to the FLU effect, NYT effects on synaptic plasticity, such as circuit switching from the cortico-spinal tract to the cortico-rubral tract, cell protection, and anti-inflammation were not observed.\u003c/p\u003e \u003cp\u003eImmunohistochemical assessment of the gastrocnemius and soleus muscles for MHC I, MHC IIa, and MHC IIb indicated that supplementary NYT to FLU mitigated MHC IIb-positive fast-twitch muscle atrophy, which is characteristically observed following ICH. Therefore, it is highly plausible that the NYT effect was primarily muscular rather than neuronal, although NYT administration alone post-ICH did not elicit any alterations in MHC I, MHC IIa, or MHC IIb expression.\u003c/p\u003e \u003cp\u003eGiven that the gastrocnemius and soleus muscles possess substantial volume and play a more significant role in maintaining the animal's body than the forelimb, the observation that additional NYT treatment of FLU following ICH resulted in atrophy prevention of the fast-twitch MHC IIb fibers may be of considerable importance in the prevention of frailty. Recent studies by other research groups have reported the effect of NYT on muscle atrophy and frailty in both normal and disease model mice, demonstrating an increase in the muscle synthesis-related factors IGF-1 and IL-6 in the blood and the promotion of mTOR and 4E-BP1 phosphorylation in the soleus muscle [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Notably, the effect of NYT on frailty and sarcopenia has recently been documented in human subjects [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Therefore, it is plausible that certain factor(s) or chemical(s) present in NYT may induce trophic factors such as IGF-1 and cytokines such as IL-6 and activate mTOR and 4E-BP in fast-twitch MHC IIb fibers.\u003c/p\u003e \u003cp\u003eAlthough the effect of FLU is relatively rapid, NYT requires a longer duration for its biological effect on ICH. Consequently, it would be valuable and necessary to investigate the weight of the gastrocnemius and soleus muscles, percentage area of muscle remaining, and percentage area of the changes in muscle subtypes at D56 in future studies, as these data were not observed following NYT administration at D28. As the mechanism in the brain after ICH might differ between FLU and NYT, the combination therapy of rehabilitative training by FLU with Kampo medicine by NYT potentially offers significant benefits for hemorrhagic stroke patients. Thus, short-term administration of FLU after ICH stimulates the reorganization of the remaining neural circuits, while long-term NYT administration affects fast-twitch myosin heavy chain IIb fibers in muscles, mitigating atrophy subsequent to ICH.\u003c/p\u003e \u003cp\u003eIn conclusion, this study investigated the efficacy of NYT for ICH and its potential additive effects when combined with rehabilitative training using FLU following ICH. The results clearly demonstrated that NYT affects ICH through a mechanism distinct from that of FLU, specifically by preventing atrophy of fast-twitch fibers post-ICH. Furthermore, the combination of FLU and NYT enhanced the effect of FLU on MDS changes on D28 and D56. These findings suggest the potential for combination therapy involving rehabilitative training and Kampo medicine to improve functional recovery following stroke.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHH was supported by a research fund from Tsumura and Co. KM was an employee of Tsumura and Co. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (23K24714 to H.H. and 24K14226 to N.T.) and by a Grant-in-Aid for Scientific Research on Innovative Areas (\u0026ldquo;Adaptive Circuit Shift\u0026rdquo; 17H05574, and \u0026ldquo;Hyper-Adaptability\u0026rdquo; 20H05476) from the Ministry of Education, Culture, Sports, Science, and Technology (to H.H.).\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCarod-Artal J, Egido JA, Gonzalez JL, Varela de Seijas E. Quality of life among stroke survivors evaluated 1 year after stroke: Experience of a stroke unit. 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Neuropeptides. 2021;90:102189. 10.1016/j.npep.2021.102189\u003cstrong\u003e\u003cstrong\u003e\u003cstrong\u003e\u003c/strong\u003e\u003c/strong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intracerebral hemorrhage, Rehabilitation, Kampo medicines, Functional recovery","lastPublishedDoi":"10.21203/rs.3.rs-5964788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5964788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRehabilitative training incorporating forced limb use (FLU) following intracerebral hemorrhagic stroke (ICH) enhances functional recovery of skilled reaching in rats. Given that Ninjin'yoeito (NYT) influences both cerebral and muscular systems, this study aimed to investigate whether the combined application of FLU and NYT could yield superior functional recovery compared to FLU alone. The ICH model was established by collagenase injection, and the subject was administered FLU from day 1 after ICH (D1) for 7 days and 1% NYT chow until D56. The combination of FLU and NYT resulted in significantly enhanced functional recovery in motor deficit scores at D28 and D56 compared with ICH only, although the score was comparable to that of the FLU group. The combination group exhibited increased total walking distance and a higher number of center entrances in the open-field test at D28. Retrograde labeling of corticospinal neurons after ICH with FluoroGold (FG) revealed no significant increase in FG-positive cells in the cortex of the combination group compared to the FLU group. Anterograde labeling with biotinylated dextran amine demonstrated increased bouton-like varicosities in the red nucleus, similar to that in the FLU group, although NYT alone did not increase the number of positive cells. Specific atrophy of MHC IIb-positive muscles after ICH was mitigated in the combination group, although no significant changes were observed in either the FLU or NYT groups. These findings indicate that the combination of FLU and NYT contributes to the functional recovery of FLU following ICH, mitigating atrophy of fast-twitch muscles.\u003c/p\u003e","manuscriptTitle":"Combining forced limb use with Ninjin'yoeito treatment prevents atrophy in fast-twitch muscles and promotes functional restoration after hemorrhagic stroke in rat models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 09:20:27","doi":"10.21203/rs.3.rs-5964788/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb9e09ca-3738-4588-a5b1-b5dd8891e3e6","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-11T17:23:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-10 09:20:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5964788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5964788","identity":"rs-5964788","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}