2-Hydroxypropyl-β-Cyclodextrin Accesses Acute and Subacute Infarcts in a Mouse Model of Ischemic Stroke

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Becktel, Elizabeth H. Le, Jennifer B. Frye, Susan A. Whitman, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7521331/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jan, 2026 Read the published version in Fluids and Barriers of the CNS → Version 1 posted 10 You are reading this latest preprint version Abstract Ischemic stroke is a leading cause of mortality and long-term disability worldwide, with limited pharmacological interventions available. 2-hydroxypropyl-β-cyclodextrin (HPβCD), a cyclic oligosaccharide approved for the treatment of Niemann-Pick disease type C, has demonstrated therapeutic potential in preclinical models of ischemic stroke by attenuating immune cell and lipid droplet accumulation in infarcts. However, the ability of HPβCD to penetrate ischemic brain tissues remains a critical determinant of its efficacy. The present study aimed to (1) assess the penetration and distribution of FITC-HPβCD within acute and subacute infarcts, which are sites of persisting blood-brain barrier (BBB) impairment, and (2) validate the accumulation of FITC-HPβCD in previously identified target organs, including the kidneys, liver, and spleen, using an aged (15-month-old) male mouse model of ischemic stroke induced by distal middle cerebral artery occlusion. We determined that FITC-HPβCD exhibits widespread systemic dissemination within 30 minutes after subcutaneous administration and is primarily eliminated via renal excretion. Notably, FITC-HPβCD selectively accumulated in the ipsilateral (i.e., infarcted) hemisphere 24 hours and 1 week after ischemic stroke, indicating that ischemia enhances the penetration of FITC-HPβCD into the brain. These results provide valuable insights into the therapeutic potential of HPβCD as a treatment for ischemic stroke and inform strategies for optimizing drug delivery to the brain in cerebrovascular diseases. Stroke Brain 2-Hydroxypropyl-β-Cyclodextrin Cyclodextrin Fluorescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Background Ischemic stroke is a leading cause of mortality and long-term disability worldwide [ 1 ]. Clinical treatment of ischemic stroke is presently limited to hyper-acute interventions that restore cerebral blood flow through intravenous thrombolysis or endovascular therapy [ 2 ], [ 3 ], [ 4 ], [ 5 ], [ 6 ], [ 7 ]. However, pharmacological interventions aimed at improving recovery and preventing neurodegeneration in the days to weeks after ischemic stroke remain unavailable, partly due to challenges in drug delivery, such as penetrating the blood-brain barrier (BBB) and achieving therapeutic concentrations in ischemic brain tissue. Importantly, the dynamic alterations in BBB permeability and tissue composition occurring throughout the acute and subacute phases of ischemic stroke are often overlooked, potentially diminishing the efficacy of candidate therapies. Understanding drug distribution and delivery during these phases is essential to develop and advance effective therapeutic interventions for ischemic stroke. 2-hydroxypropyl-β-cyclodextrin (HPβCD), a cyclic oligosaccharide comprised of glucose monomers that form a hydrophobic interior and hydrophilic exterior, is approved by the U.S. Food and Drug Administration (FDA) for use as an inert excipient in pharmaceutical formulations and has, therefore, undergone extensive safety and toxicity studies [ 8 ]. HPβCD is currently under investigation as a potential treatment for Niemann-Pick disease type C (NPC); however, it has not yet received FDA approval for this indication. In the context of NPC, a neurodegenerative disorder characterized by the endolysosomal accumulation of unesterified cholesterol and sphingolipids, HPβCD has been shown to delay the onset of clinical symptoms and reduce accumulation of cholesterol and gangliosides in neuronal cells [ 9 ]. Additionally, HPβCD has proven efficacious in models of atherosclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [ 10 ], [ 11 ], [ 12 ]. HPβCD has also been investigated as an active pharmaceutical ingredient (API) for the prospective treatment of ischemic stroke. We previously demonstrated that repeated administration of HPβCD in young adult and aged mouse models attenuates immune cell infiltration and lipid droplet accumulation in infarcts and improves recovery at transcriptional and functional levels 7 weeks after ischemic stroke [ 13 ]. Furthermore, we determined that HPβCD induces liver X receptor (LXR)-mediated transcriptional reprogramming in mouse stroke infarcts – a mechanism that aligns with the HPβCD-induced activation of LXR target genes observed in models of atherosclerotic plaque [ 11 ], [ 13 ]. However, the efficacy of HPβCD as a treatment for ischemic stroke is dependent on the ability of the therapeutic agent to penetrate ischemic brain tissues, a process influenced by dynamic alterations in BBB integrity and tissue composition over time. The present study aimed to (1) assess the penetration and distribution of FITC-HPβCD within acute and subacute stroke infarcts, which are sites of persisting BBB impairment, and (2) validate the accumulation of FITC-HPβCD in previously identified target organs, including the kidneys, liver, and spleen. Using advanced imaging and analytical techniques, we elucidated the extent of FITC-HPβCD delivery to ischemic brain and peripheral tissues in aged (15-month-old) male mice subjected to stroke induced by distal middle cerebral artery occlusion 24 hours or 1 week prior to subcutaneous administration of FITC-HPβCD. In characterizing the spatiotemporal dynamics of FITC-HPβCD penetration, we seek to provide valuable insights into the potential of HPβCD as a pharmacological intervention for ischemic stroke and inform the development of strategies for enhanced drug delivery to the brain in cerebrovascular diseases. 2. Methods 2.1. Materials 6-deoxy-6-[(5/6)-fluoresceinylthioureido]-(2-hydroxypropyl)-β-cyclodextrin (FITC-HPβCD) was purchased from CarboHyde Zrt. (Identification No. CH-0006). The average degrees of substitution determined by NMR were 1.0 for FITC and 4.5 for (2-hydroxypropyl) groups. FITC-HPβCD underwent extensive dialysis to remove free dye content and 6-deoxy-6-monoamino-HPβCD impurities. FITC-HPβCD, in powder form, was stored at 4°C in a tightly sealed dark glass container to protect from direct light and heat. 2.2. Animals Aged (15-month-old) wild-type male C57BL/6J (Stock No. 000664) mice were purchased from The Jackson Laboratory. Mice were housed in a temperature-controlled suite with a 12-hour light-dark regimen and ad libitum access to food and water. All experimental procedures were conducted in accordance with the animal care standards of the National Institutes of Health and approved by the University of Arizona Institutional Animal Care and Use Committee. 2.3. Stroke Surgeries Stroke was induced in mice using the distal middle cerebral artery occlusion + hypoxia (DH) model. The DH stroke model generates a sizable infarct (24% of the ipsilateral hemisphere centered on the somatosensory cortex), has low variability, and has exceptional long-term survivability. The addition of hypoxia is necessary because C57BL/6 mice that undergo DH stroke without hypoxia have much smaller infarcts. The methodology of the DH stroke model and validation of the controls has been previously published [ 14 ], [ 15 ]. To induce stroke, mice were anesthetized by isoflurane inhalation and maintained at 37°C throughout the surgical procedure. For all experiments, mice were injected subcutaneously (s.c.) with a single dose of buprenorphine hydrochloride (0.1 mg/kg) dissolved in sterile saline. Following pre-operative preparation, an incision was made to expose the right temporoparietal skull between the orbit and the ear. Under an operating microscope, a small hole was made with a high-speed microdrill through the outer surface of the semi-translucent skull over the visually identified middle cerebral artery (MCA) at the level of the parietal cerebral artery. Permanent occlusion of the MCA was performed by electrocoagulation with a small vessel cauterizer. Surgical wounds were closed using Surgi-lock 2oc tissue adhesive. Mice were then immediately transferred to a hypoxia chamber containing 9% oxygen and 91% nitrogen for 45 minutes. Extended-release buprenorphine (3.25 mg/kg Ethiqa XR, s.c.) was administered 24 hours after surgery as post-operative analgesia. 2.4. MRI Infarct volumes were assessed by MRI 24 hours after stroke using a Bruker BioSpec 70/20 7.0T scanner with ParaVision-360.3.2 software and a 4-channel phase array mouse coil. Mice were placed in a cradle equipped with a stereotaxic frame, an integrated heating system to maintain body temperature at 37 ± 1°C, and a pressure probe to monitor respiration. During MRI acquisition, anesthesia was maintained by inhalation of 1.5-3% isoflurane. High-resolution structural images were acquired using a T 2 -weighted RARE Bruker pulse sequence with the following parameters: repetition time ( TR ) = 2500 ms; flip angle = 30°; RARE factor = 8; matrix size = 256 x 256; averages = 2; field of view = 20 mm x 20 mm; slice thickness = 0.8 mm; number of slices = 15; acquisition time = 2 minutes 40 seconds. Infarcts and hemispheric cross sections were manually delineated on T 2 -weighted MR images using Mango v4.1. 2.5. In Vivo and Ex Vivo Fluorescence Imaging At 24 hours or 1 week after stroke, 3.5 mg FITC-HPβCD dissolved in 0.2 mL sterile saline (100 mg/kg FITC-HPβCD, s.c.) was injected into the hip region of each mouse. Mice were anesthetized by isoflurane inhalation and placed in a prone position on the imaging platform of a Spectral Instruments Imaging Lago X. At 0, 5, 10, 15, 20, 25, and 30 minutes after injection of FITC-HPβCD, in vivo fluorescence images were captured (𝜆 ex : 465 nm; 𝜆 em : 510 nm). After 30 minutes, perfused organs were dissected for ex vivo imaging. Fluorescence signal strength was quantified using Spectral Instruments Imaging Aura v4.0. 2.6. Plasma Collection At 1 and 15 minutes after injection of FITC-HPβCD, a heparinized microhematocrit capillary tube was gently inserted into the retro-orbital sinus, positioned at the medial canthus of the eye. Blood was collected by capillary action into an EDTA-coated microcentrifuge tube. At 30 minutes after injection of FITC-HPβCD, mice were secured in a supine position. The thoracic cavity of each mouse was sterilized with 70% ethanol, and a midline incision was performed to expose the heart. A sterile syringe, equipped with a fine-gauge needle, was inserted into the left ventricle of the heart, and blood was drawn by gentle aspiration. Blood was immediately expelled into an EDTA-coated microcentrifuge tube. Collected blood samples were incubated for 1 hour at 37°C then centrifuged at 4°C for 10 minutes at 5000 x g to separate blood plasma. Plasma samples were stored at -80°C until further analysis. 2.7. Tissue Collection Following exsanguination by intracardiac bleed, mice were perfused with warm 0.9% saline. Organs (brain, heart, liver, spleen, and kidney) were then dissected for ex vivo imaging. Upon completion of ex vivo imaging, organs were prepared for tissue processing. To obtain tissue samples from the heart, liver, spleen, and kidney, smaller pieces of each organ were isolated and crushed with forceps. To obtain tissue samples from the brain, the olfactory bulb and cerebellum were first removed using a brain mold and razor blade. The brain was then divided into contralateral and ipsilateral hemispheres down the longitudinal fissure and stored separately. All tissue samples were flash frozen in liquid nitrogen and stored at -80°C until further analysis. 2.8. Tissue Processing Tissue samples were processed prior to total protein quantification in batches of 10–15 samples. Samples were placed on ice and allowed to thaw for 5 minutes. 500 µL of radioimmunoprecipitation assay (RIPA) buffer was added to heart, liver, spleen, and kidney samples, and 1 mL of RIPA buffer was added to brain samples. Samples were sonicated then centrifuged at 4°C for 15 minutes at 13,000 x g . Supernatant was collected from each sample and placed in a 1.5 mL Eppendorf tube. Total protein quantification was performed using a Millipore Direct Detect Spectrometer according to manufacturer instructions. FITC-HPβCD content of each sample was normalized to tissue protein content. 2.9. Microplate Reader Quantification of FITC-HPβCD Aliquots of plasma and tissue samples were placed into black 96-well plates, and fluorescence intensities were measured using a BioTek Cytation 3 Microplate Reader according to manufacturer instructions (𝜆 ex : 492 nm; 𝜆 em : 520 nm). Each standard, experimental plasma sample, and experimental tissue sample was measured in duplicate. The lower limit of detection for FITC-HPβCD in mouse plasma and tissue samples was 0.01 µg/mL. Fluorescence intensities were quantified using BioTek Gen5 v2.06. 2.10. Statistical Analysis Statistical analyses were performed using GraphPad Prism v10.2.3. Data are expressed as mean ± SD. Differences were considered significant at P < 0.05. Full statistical results, including the statistical tests, number of observations, test statistics, exact probabilities, and degrees of freedom, are reported in Supplementary Table 1 . 3. Results 3.1. In vivo imaging of FITC-HPβCD in aged mice 24 hours after stroke To assess the accumulation and distribution of FITC-HPβCD in the acute and subacute phases after stroke, aged (15-month-old) male mice were subjected to DH stroke. At 24 hours after stroke, T 2 -weighted MRI was performed to confirm infarct generation. At 24 hours or 1 week after stroke, mice were injected with FITC-HPβCD (100 mg/kg, s.c.) and immediately placed in a Spectral Instruments Imaging Lago X for in vivo imaging. At 0, 5, 10, 15, 20, 25, and 30 minutes after injection of FITC-HPβCD, serial images of mice were captured. At 1, 15, and 30 minutes after injection of FITC-HPβCD, plasma was collected for fluorescence quantification on a microplate reader. Upon completion of in vivo imaging and subsequent plasma collection at 30 minutes after injection of FITC-HPβCD, mice were perfused with warm saline, and organs (i.e., heart, brain, liver, spleen, and kidney) were dissected for ex vivo imaging and subsequent fluorescence quantification on a microplate reader ( Fig. 1 A ) . At 24 hours after stroke, T 2 -weighted MRI was performed to confirm infarct generation and ensure proper stratification of mice across experimental groups. The quantification of T 2 -weighted MRI scans verified that acute infarct volume did not differ significantly between groups ( Fig. 1 B ) . Upon completion of T 2 -weighted MRI, mice were injected with FITC-HPβCD (100 mg/kg, s.c.) and immediately placed in a Spectral Instruments Imaging Lago X for in vivo imaging. The quantification of serial in vivo images revealed increasing fluorescence intensities over 30 minutes in mice injected with FITC-HPβCD. Notably, FITC-HPβCD disseminated comparably throughout the bodies of both naïve mice and those subjected to DH stroke 24 hours prior. Additionally, minimal fluorescence was observed in uninjected naïve mice and those subjected to DH stroke 24 hours prior ( Fig. 1 C-D ) . 3.2. Ex vivo imaging of FITC-HPβCD in organs from aged mice 24 hours after stroke At 30 minutes after injection of FITC-HPβCD, organs (i.e., heart, brain, liver, spleen, and kidney) were dissected for ex vivo imaging. The quantification of ex vivo images revealed that FITC-HPβCD can permeate the brain 24 hours after DH stroke; FITC-HPβCD localized primarily to the ipsilateral (i.e., infarcted) hemisphere ( Fig. 2 A-B ) . Importantly, FITC-HPβCD was processed similarly in the livers and kidneys of both naïve mice and those subjected to DH stroke 24 hours prior; however, FITC-HPβCD accumulated to a greater extent in the brains of mice subjected to DH stroke compared to brains of naïve mice. Additionally, minimal fluorescence was detected in the spleens and hearts of uninjected and injected mice ( Fig. 2 C-D ) . 3.3. FITC-HPβCD concentrations in organs from aged mice 24 hours after stroke Upon completion of ex vivo imaging, organs were processed for subsequent fluorescence quantification on a microplate reader. FITC-HPβCD was predominantly localized to the kidneys 30 minutes after injection in naïve mice and those subjected to DH stroke 24 hours prior ( Fig. 3 A ) . However, FITC-HPβCD was also detected in the livers and hearts of naïve mice and those subjected to DH stroke 24 hours prior. Interestingly, FITC-HPβCD accumulated in the spleens of mice subjected to DH stroke 24 hours prior but did not accumulate significantly in the spleens of naïve mice ( Fig. 3 B ) . Additionally, FITC-HPβCD was concentrated in the ipsilateral (i.e., infarcted) hemispheres compared to the corresponding contralateral hemispheres of brains dissected from mice subjected to DH stroke 24 hours prior ( Fig. 3 C ) . FITC-HPβCD also temporally accumulated in the plasma of naïve mice and mice subjected to DH stroke 24 hours prior ( Fig. 3 D ) . 3.4. In vivo imaging of FITC-HPβCD in aged mice 1 week after stroke To assess the accumulation and distribution of FITC-HPβCD in the subacute phase after stroke, aged (15-month-old) male mice were subjected to DH stroke 1 week prior to injection of FITC-HPβCD. The quantification of serial in vivo images revealed increasing fluorescence intensities over 30 minutes in mice injected with FITC-HPβCD. Analogous to the distribution of FITC-HPβCD in the acute phase after stroke, FITC-HPβCD disseminated comparably throughout the bodies of both naïve mice and those subjected to DH stroke 1 week prior. Additionally, minimal fluorescence was observed in uninjected naïve mice and those subjected to DH stroke 1 week prior ( Fig. 4 A-B ) . 3.5. Ex vivo imaging of FITC-HPβCD in organs from aged mice 1 week after stroke At 30 minutes after injection of FITC-HPβCD, organs (i.e., heart, brain, liver, spleen, and kidney) were dissected for ex vivo imaging. The quantification of ex vivo images revealed that FITC-HPβCD can permeate the brain 1 week after DH stroke; FITC-HPβCD localized primarily to the ipsilateral (i.e., infarcted) hemisphere. Notably, subacute stroke infarcts in uninjected mice exhibited green-channel autofluorescence (GCAF), consistent with prior observations in mouse models of ischemic stroke [ 16 ]. GCAF can be attributed to the intracellular accumulation of lipofuscin, an autofluorescent lipid-rich polymeric entity, caused by oxidative stress [ 17 ] ( Fig. 5 A-B ) . FITC-HPβCD accumulated similarly in the livers, kidneys, and hearts of both naïve mice and those subjected to DH stroke 1 week prior; however, FITC-HPβCD accumulated to a greater extent in the brains of mice subjected to DH stroke compared to brains of naïve mice. Interestingly, FITC-HPβCD was detected in the spleens of mice subjected to DH stroke 1 week prior when compared to uninjected naïve mice ( Fig. 5 C-D ) . 3.6. FITC-HPβCD concentrations in organs from aged mice 1 week after stroke Upon completion of ex vivo imaging, organs were processed for subsequent fluorescence quantification on a microplate reader. FITC-HPβCD was predominantly localized to the kidneys 30 minutes after injection in naïve mice and those subjected to DH stroke 1 week prior ( Fig. 6 A ) . However, FITC-HPβCD was also detected in the livers, spleens, and hearts of naïve mice and those subjected to DH stroke 1 week prior ( Fig. 6 B ) . Additionally, FITC-HPβCD was concentrated in the ipsilateral (i.e., infarcted) hemispheres compared to the corresponding contralateral hemispheres of brains dissected from mice subjected to DH stroke 1 week prior ( Fig. 6 C ) . FITC-HPβCD also temporally accumulated in the plasma of naïve mice and mice subjected to DH stroke 1 week prior ( Fig. 6 D ) . 4. Discussion HPβCD, a chemically modified derivative of β-cyclodextrin, has emerged as a versatile molecule with numerous applications in the pharmaceutical sector. Initially patented as an excipient nearly forty years ago, HPβCD is commonly utilized to enhance the aqueous solubility, stability, and bioavailability of steroids, antivirals, and chemotherapies [ 18 ], [ 19 ]. HPβCD has also more recently been investigated as an API. These investigations were prompted by its widely approved safety profile and capacity to interact with biomolecules, such as cholesterol. HPβCD is included in both the European Pharmacopoeia (PhEur) and United States Pharmacopoeia (USP) and received an orphan drug designation for the treatment of NPC, although it has not yet received FDA approval for this indication. Currently, research is ongoing to evaluate the therapeutic efficacy of HPβCD in related diseases, including atherosclerosis, Alzheimer’s disease, Parkinson’s disease, and ischemic stroke, all of which exhibit pathological accumulation of aggregation-prone peptides, proteins, or lipids in various tissues [ 11 ], [ 12 ], [ 13 ], [ 20 ], [ 21 ]. Extensive investigations into the toxicity, metabolism, and pharmacokinetics of HPβCD in humans and animals have consistently demonstrated that it is well tolerated across species [ 22 ]. These studies have determined that parenteral (e.g., intravenous and subcutaneous) administration of HPβCD is characterized by rapid distribution within the extracellular fluid compartment due to its hydrophilicity [ 23 ]. HPβCD is not significantly metabolized by the liver or other tissues and is primarily eliminated intact in the urine via renal excretion. In humans, the plasma half-life after intravenous administration is relatively short, ranging from 1 to 2 hours [ 24 ]. Additionally, HPβCD does not readily cross lipid membranes, including the BBB, unless administered intrathecally [ 25 ], [ 26 ]. In the present study, we aimed to (1) assess the penetration and distribution of FITC-HPβCD within acute and subacute stroke infarcts, which are sites of persisting BBB impairment, and (2) validate the accumulation of FITC-HPβCD in previously identified target organs, including the kidneys, liver, and spleen. Gould and Scott (2005) previously reviewed several toxicology studies indicating that intravenous administration of HPβCD affected main target organs, including the kidneys, liver, lungs, and spleen [ 22 ]. In accordance with published human and animal studies, we determined that FITC-HPβCD was predominantly localized to the kidneys 30 minutes after subcutaneous administration in both naïve mice and those subjected to DH stroke 24 hours or 1 week prior, further substantiating renal excretion as the primary route of elimination. Importantly, Váradi et al. reported a comparable accumulation of FITC-HPβCD in the kidneys of male BALB/c mice 60 minutes after intravenous administration [ 27 ]. In addition to the kidneys, FITC-HPβCD was detected in the livers and, to a lesser extent, in the hearts and spleens of both naïve mice and those subjected to DH stroke 24 hours or 1 week prior. It is plausible that the modest concentrations of FITC-HPβCD observed in the hearts and spleens may be attributed to its endocytic uptake by cardiac and splenic endothelial cells, a phenomenon previously described in human umbilical vein endothelial cells (HUVECs) [ 27 ]. These results indicate that HPβCD is distributed and excreted comparably after subcutaneous and intravenous administration and provide valuable insights into the pharmacokinetics and tissue-specific accumulation of HPβCD during the acute and subacute phases of ischemic stroke recovery. FITC-HPβCD also selectively accumulated in the ipsilateral (i.e., infarcted) hemispheres compared to the contralateral hemispheres of brains at 24 hours or 1 week after DH stroke. The preferential accumulation of FITC-HPβCD in the ipsilateral (i.e., infarcted) hemisphere reflects the increased permeability of the BBB in the affected region(s), a recognized hallmark of ischemic stroke [ 28 ]. These results are particularly relevant to the potential application of HPβCD in facilitating the clearance of cholesterol and other lipids implicated in ischemic stroke pathology, which our previous studies indicate drive secondary neurodegeneration through inflammatory mechanisms [ 13 ], [ 29 ]. However, there are several methodological limitations that must be acknowledged. Firstly, these imaging and analytical analyses were conducted exclusively in aged (15-month-old) male C57BL/6J mice. We recognize the necessity of repeating these analyses, particularly the in vivo imaging, in male and female BALB/c mice due to their lighter coat color, which would allow for improved visualization of FITC-HPβCD. However, Váradi et al. reported that FITC-HPβCD produced a potent fluorescent signal in the highly perfused skin capillaries of BALB/c mice, which concealed fluorescent signals from specific internal organs [ 27 ]. Secondly, we opted to administer a single dose of 100 mg/kg FITC-HPβCD via subcutaneous injection 24 hours or 1 week after DH stroke. Future studies should incorporate the following: (1) therapeutic concentrations of FITC-HPβCD ranging from 100 mg/kg to 4000 mg/kg, (2) intravenous or intraperitoneal routes of administration, (3) models of ischemic stroke involving reperfusion (e.g., intraluminal filament MCA occlusion), and (4) additional end points ranging from 2 weeks to 8 weeks after ischemic stroke. In conclusion, the present study bolsters the potential of HPβCD as an API for the treatment of ischemic stroke. The observed pharmacokinetic profile, characterized by renal excretion as the primary route of elimination and selective accumulation in infarcted brain regions with compromised BBB integrity, underscores its capacity to facilitate the clearance of cholesterol and other lipid byproducts resulting from myelin and cellular membrane degradation after ischemic stroke. Combined with its well-established safety profile, these results provide a strong rationale for further investigation into the therapeutic applications of HPβCD. Declarations Ethics Approval and Consent to Participate :All experimental procedures were conducted in accordance with the animal care standards of the National Institutes of Health and approved by the University of Arizona Institutional Animal Care and Use Committee. Consent for Publication : Not applicable. Availability of Data and Materials : The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing Interests : The authors declare that they have no competing interests. Funding : This research was funded by the National Institute of Neurological Disorders and Stroke RF1NS131110 (K.D.), National Institute on Aging R01AG063808 (K.D.), United States Department of Veterans Affairs I01RX003224 (R.S.), National Institute on Aging T32AG058503-01A1 (D.B.), and the Fondation Leducq Transatlantic Network of Excellence Stroke-IMPaCT (K.D.). Authors’ Contributions : Conceptualization, D.B. and K.D.; Methodology, D.B., J.F., S.W. and K.D.; Formal Analysis, D.B., E.L., J.F. and S.W.; Investigation, D.B., E.L., J.F. and S.W.; Resources, K.D.; Writing – Original Draft, D.B. and E.L.; Writing – Review & Editing, R.S. and K.D.; Visualization, D.B., E.L. and K.D.; Supervision, K.D.; Project Administration, D.B. and K.D.; Funding Acquisition, R.S. and K.D. Acknowledgments : We thank Dr. Milo Malanga at CarboHyde Zrt. for his efforts in the synthesis and characterization of FITC-HPβCD. References Martin SS et al. 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J Inherit Metab Dis. 2020;43. 10.1002/jimd.12189 . Ory DS et al. Oct., ‘Intrathecal 2-hydroxypropyl-β-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1–2 trial’, The Lancet , vol. 390, no. 10104, pp. 1758–1768, 2017, 10.1016/S0140-6736(17)31465-4 Váradi J et al. Oct., ‘Pharmacokinetic Properties of Fluorescently Labelled Hydroxypropyl-Beta-Cyclodextrin’, Biomolecules , vol. 9, no. 10, 2019, 10.3390/BIOM9100509 Zbesko JC, et al. Glial scars are permeable to the neurotoxic environment of chronic stroke infarcts. Neurobiol Dis. 2018. 10.1016/j.nbd.2018.01.007 . Chung AG et al. ‘Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis and mediates secondary neurodegeneration in an osteopontin-dependent mechanism’, eNeuro , vol. 5, no. 5, 2018, 10.1523/ENEURO.0076-18.2018 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1StatisticalTests.xlsx Cite Share Download PDF Status: Published Journal Publication published 28 Jan, 2026 Read the published version in Fluids and Barriers of the CNS → Version 1 posted Editorial decision: Revision requested 03 Nov, 2025 Reviews received at journal 22 Oct, 2025 Reviews received at journal 17 Sep, 2025 Reviewers agreed at journal 11 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers invited by journal 07 Sep, 2025 Editor assigned by journal 04 Sep, 2025 Submission checks completed at journal 04 Sep, 2025 First submitted to journal 02 Sep, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7521331","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513796137,"identity":"df3c9c6a-0cf8-4612-8149-0b8feb1a210f","order_by":0,"name":"Danielle A. Becktel","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Danielle","middleName":"A.","lastName":"Becktel","suffix":""},{"id":513796138,"identity":"3b38b92b-806c-4dfa-b40a-9c306b90f51f","order_by":1,"name":"Elizabeth H. Le","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"H.","lastName":"Le","suffix":""},{"id":513796139,"identity":"4bd0bd11-9ddb-4b5b-95b1-5ddd9ed345d3","order_by":2,"name":"Jennifer B. Frye","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"B.","lastName":"Frye","suffix":""},{"id":513796140,"identity":"800f7039-0a85-4c4f-96e1-44155d00a5e5","order_by":3,"name":"Susan A. Whitman","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"A.","lastName":"Whitman","suffix":""},{"id":513796141,"identity":"cd24ccb6-7b15-437b-a570-84067de399ac","order_by":4,"name":"Rick G. Schnellmann","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Rick","middleName":"G.","lastName":"Schnellmann","suffix":""},{"id":513796142,"identity":"b15c7f47-a648-4d30-95cd-61d0856b14b0","order_by":5,"name":"Kristian P. Doyle","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYDCCAwyMB4CEHAMDYwNUKIGgFgaQFmPStSQ2IIQIaOE73mNw4MOZe+n9sw83f/i4w46Bnz3HAK8WyTNnDA7OuFGcO+NcYoPhzDPJDJI9b/BrMbiRY3CY50NCbsMZxoZk3jZmsAhhLX8+JKTLA7Uc5m2rZ7AnSgvDjYQEgzOMjc28bYcZDCQI+uVYwcGeMwmGG88wNjPObDvOI3HmWQFeLXzHmzc++HEsQV7uDPvjDx/bquX425M34NXCwMCB6gweAspBgP0BEYpGwSgYBaNgRAMAnCpS91XZ65MAAAAASUVORK5CYII=","orcid":"","institution":"University of Arizona","correspondingAuthor":true,"prefix":"","firstName":"Kristian","middleName":"P.","lastName":"Doyle","suffix":""}],"badges":[],"createdAt":"2025-09-02 22:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7521331/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7521331/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12987-026-00767-9","type":"published","date":"2026-01-28T15:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91211304,"identity":"3650d1a6-074c-42e6-8419-2452311e2443","added_by":"auto","created_at":"2025-09-12 17:57:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":684332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo imaging of FITC-HPβCD in aged mice 24 hours after stroke. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eExperimental design, 15-month-old male mice were subjected to distal middle cerebral artery occlusion + hypoxia (DH) stroke.\u003cem\u003e T\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MRI was performed 24 hours after DH stroke to confirm infarct generation. At 24 hours or 1 week after stroke, mice were injected with FITC-HPβCD (100 mg/kg, s.c.) and immediately placed in a Spectral Instruments Imaging Lago X for in vivo imaging. At 0, 5, 10, 15, 20, 25, and 30 minutes after injection of FITC-HPβCD, serial images of mice were captured. At 1, 15, and 30 minutes after injection of FITC-HPβCD, plasma was collected for fluorescence quantification on a microplate reader. Upon completion of in vivo imaging and subsequent plasma collection at 30 minutes, mice were perfused with warm saline, and organs (i.e., heart, brain, liver, spleen, and kidney) were dissected for ex vivo imaging and subsequent fluorescence quantification on a microplate reader.\u003cem\u003e\u003cstrong\u003e B, \u003c/strong\u003e\u003c/em\u003eQuantification of\u003cem\u003e T\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MRI scans obtained 24 hours after DH stroke confirmed that acute infarct volume did not differ significantly between groups (\u003cem\u003en\u003c/em\u003e = 3-8; ordinary one-way ANOVA; Tukey’s multiple comparisons test, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Representative \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MRI scans acquired 24 hours after DH stroke depict infarcts primarily localized to the somatosensory cortices. \u003cem\u003e\u003cstrong\u003eC, \u003c/strong\u003e\u003c/em\u003eRepresentative in vivo images captured at 5, 10, 15, 20, 25, and 30 minutes after injection of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 465 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 510 nm). Background fluorescence was manually subtracted using Spectral Instruments Imaging Aura v4.0. \u003cem\u003e\u003cstrong\u003eD, \u003c/strong\u003e\u003c/em\u003eQuantification of serial in vivo images revealed increasing fluorescence intensities over time in mice injected with FITC-HPβCD (\u003cem\u003en\u003c/em\u003e = 5-8; RM two-way ANOVA with the Geisser-Greenhouse correction; Dunnett’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/a7211b5b7205df4b357518b7.png"},{"id":91210725,"identity":"9b710ab5-5e15-495a-afca-9def19b60fc0","added_by":"auto","created_at":"2025-09-12 17:49:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":404359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivo imaging of FITC-HPβCD in organs from aged mice 24 hours after stroke. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eRepresentative ex vivo images of brains dissected at 30 minutes after injection of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 465 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 510 nm). Background fluorescence was manually subtracted using Spectral Instruments Imaging Aura v4.0. \u003cem\u003e\u003cstrong\u003eB, \u003c/strong\u003e\u003c/em\u003eQuantification of ex vivo images revealed that FITC-HPβCD can permeate the brain after stroke (\u003cem\u003en\u003c/em\u003e = 4-7; ordinary one-way ANOVA; Tukey’s multiple comparisons test, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001). \u003cem\u003e\u003cstrong\u003eC, \u003c/strong\u003e\u003c/em\u003eRepresentative ex vivo images of organs (i.e., livers, kidneys, spleens, hearts, and brains) dissected 30 minutes after injection of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 465 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 510 nm). Background fluorescence was manually subtracted using Spectral Instruments Imaging Aura v4.0. \u003cem\u003e\u003cstrong\u003eD, \u003c/strong\u003e\u003c/em\u003eQuantification of ex vivo images revealed that FITC-HPβCD accumulates in the livers and kidneys of both naïve mice and those subjected to stroke; however, FITC-HPβCD accumulates to a greater extent in the brains of mice subjected to stroke (\u003cem\u003en \u003c/em\u003e= 5-8; ordinary two-way ANOVA; Tukey’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/ee89ee62f5a92351b2c58ce4.png"},{"id":91210728,"identity":"ccfd8534-11ed-49fd-9771-da47fab32808","added_by":"auto","created_at":"2025-09-12 17:49:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":348121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFITC-HPβCD concentrations in organs from aged mice 24 hours after stroke. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA, \u003c/strong\u003e\u003c/em\u003eMicroplate reader quantification of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 492 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 520 nm) in tissue samples demonstrated that FITC-HPβCD was predominantly localized to the kidneys 30 minutes after injection (\u003cem\u003en \u003c/em\u003e= 5-9; ordinary two-way ANOVA; Tukey’s multiple comparisons test, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001). \u003cem\u003e\u003cstrong\u003eB, \u003c/strong\u003e\u003c/em\u003eStatistical analysis of each set of organs (i.e., livers, kidneys, spleens, and hearts) revealed that FITC-HPβCD accumulates in the livers, kidneys, and hearts of both naïve mice and those subjected to stroke; however, FITC-HPβCD accumulates to a greater extent in the spleens of mice subjected to stroke compared to naïve mice (\u003cem\u003en \u003c/em\u003e= 5-9; ordinary one-way ANOVA; Tukey’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001). \u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e, Statistical analysis of brain hemispheres demonstrated that FITC-HPβCD predominantly accumulates in the ipsilateral (i.e., infarcted) hemispheres of mice subjected to stroke (\u003cem\u003en \u003c/em\u003e= 5-9; ordinary one-way ANOVA; Tukey’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001). \u003cem\u003e\u003cstrong\u003eD,\u003c/strong\u003e\u003c/em\u003e Microplate reader quantification of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 492 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 520 nm) in plasma samples validated that FITC-HPβCD temporally accumulates in the circulation within 30 minutes after injection (\u003cem\u003en \u003c/em\u003e= 3-9; ordinary one-way ANOVA; Tukey’s multiple comparisons test, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/55a788071b5e0ef8d2102028.png"},{"id":91210730,"identity":"2215c377-49ff-4b67-b9fe-13beb7818f90","added_by":"auto","created_at":"2025-09-12 17:49:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":649342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo imaging of FITC-HPβCD in aged mice 1 week after stroke.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e A, \u003c/strong\u003e\u003c/em\u003eRepresentative in vivo images captured at 5, 10, 15, 20, 25, and 30 minutes after injection of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 465 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 510 nm). Background fluorescence was manually subtracted using Spectral Instruments Imaging Aura v4.0.\u003cem\u003e\u003cstrong\u003e B, \u003c/strong\u003e\u003c/em\u003eQuantification of serial in vivo images revealed increasing fluorescence intensities over time in mice injected with FITC-HPβCD (\u003cem\u003en\u003c/em\u003e = 4; RM two-way ANOVA with the Geisser-Greenhouse correction; Dunnett’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/e432ad410ee67649287970f6.png"},{"id":91210734,"identity":"1a282485-c614-4c85-8cb0-e65fa2ae8951","added_by":"auto","created_at":"2025-09-12 17:49:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":404761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivo imaging of FITC-HPβCD in organs from aged mice 1 week after stroke.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e A,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eRepresentative ex vivo images of brains dissected at 30 minutes after injection of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 465 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 510 nm). Background fluorescence was manually subtracted using Spectral Instruments Imaging Aura v4.0. \u003cem\u003e\u003cstrong\u003eB, \u003c/strong\u003e\u003c/em\u003eQuantification of ex vivo images revealed that FITC-HPβCD can permeate the brain after stroke (\u003cem\u003en\u003c/em\u003e = 4; ordinary one-way ANOVA; Tukey’s multiple comparisons test, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). \u003cem\u003e\u003cstrong\u003eC, \u003c/strong\u003e\u003c/em\u003eRepresentative ex vivo images of organs (i.e., livers, kidneys, spleens, hearts, and brains) dissected 30 minutes after injection of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 465 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 510 nm). Background fluorescence was manually subtracted using Spectral Instruments Imaging Aura v4.0. \u003cem\u003e\u003cstrong\u003eD, \u003c/strong\u003e\u003c/em\u003eQuantification of ex vivo images revealed that FITC-HPβCD accumulates in the livers, kidneys, and hearts of both naïve mice and those subjected to stroke; however, FITC-HPβCD accumulates to a greater extent in the brains and spleens of mice subjected to stroke (\u003cem\u003en \u003c/em\u003e= 4; ordinary two-way ANOVA; Tukey’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/ebf92a4d46b79c990bf1dacf.png"},{"id":91210733,"identity":"2e42b047-5a59-4d67-a50e-1c21d0afce57","added_by":"auto","created_at":"2025-09-12 17:49:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":343275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFITC-HPβCD concentrations in organs from aged mice 1 week after stroke. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA, \u003c/strong\u003e\u003c/em\u003eMicroplate reader quantification of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 492 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 520 nm) in tissue samples demonstrated that FITC-HPβCD was predominantly localized to the kidneys 30 minutes after injection (\u003cem\u003en \u003c/em\u003e= 3-9; ordinary two-way ANOVA; Tukey’s multiple comparisons test, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001). \u003cem\u003e\u003cstrong\u003eB, \u003c/strong\u003e\u003c/em\u003eStatistical analysis of each set of organs (i.e., livers, spleens, kidneys, and hearts) revealed that FITC-HPβCD accumulates in the livers, spleens, kidneys, and hearts of both naïve mice and those subjected to stroke (\u003cem\u003en \u003c/em\u003e= 3-9; ordinary one-way ANOVA; Tukey’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). \u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e, Statistical analysis of brain hemispheres demonstrated that FITC-HPβCD predominantly accumulates in the ipsilateral (i.e., infarcted) hemispheres of mice subjected to stroke (\u003cem\u003en \u003c/em\u003e= 3-9; ordinary one-way ANOVA; Tukey’s multiple comparisons test, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001). \u003cem\u003e\u003cstrong\u003eD,\u003c/strong\u003e\u003c/em\u003e Microplate reader quantification of FITC-HPβCD (𝜆\u003csub\u003eex\u003c/sub\u003e: 492 nm; 𝜆\u003csub\u003eem\u003c/sub\u003e: 520 nm) in plasma samples validated that FITC-HPβCD temporally accumulates in the circulation within 30 minutes after injection (\u003cem\u003en \u003c/em\u003e= 3-9; ordinary one-way ANOVA; Tukey’s multiple comparisons test, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/c92b17c7be2dd0eed35296cf.png"},{"id":101691250,"identity":"a236b2fc-9585-4de0-bf7d-c3c30cd903f8","added_by":"auto","created_at":"2026-02-02 16:13:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3886322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/eb9a15a4-2ce3-4532-a52a-81564121a1dd.pdf"},{"id":91210726,"identity":"1bc10006-b4a3-4981-93fa-fe5dfdfb3b4e","added_by":"auto","created_at":"2025-09-12 17:49:41","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22248,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1StatisticalTests.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7521331/v1/afbe57306b8ea1f394502d6e.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"2-Hydroxypropyl-β-Cyclodextrin Accesses Acute and Subacute Infarcts in a Mouse Model of Ischemic Stroke","fulltext":[{"header":"1. Background","content":"\u003cp\u003eIschemic stroke is a leading cause of mortality and long-term disability worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Clinical treatment of ischemic stroke is presently limited to hyper-acute interventions that restore cerebral blood flow through intravenous thrombolysis or endovascular therapy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, pharmacological interventions aimed at improving recovery and preventing neurodegeneration in the days to weeks after ischemic stroke remain unavailable, partly due to challenges in drug delivery, such as penetrating the blood-brain barrier (BBB) and achieving therapeutic concentrations in ischemic brain tissue. Importantly, the dynamic alterations in BBB permeability and tissue composition occurring throughout the acute and subacute phases of ischemic stroke are often overlooked, potentially diminishing the efficacy of candidate therapies. Understanding drug distribution and delivery during these phases is essential to develop and advance effective therapeutic interventions for ischemic stroke.\u003c/p\u003e\u003cp\u003e2-hydroxypropyl-β-cyclodextrin (HPβCD), a cyclic oligosaccharide comprised of glucose monomers that form a hydrophobic interior and hydrophilic exterior, is approved by the U.S. Food and Drug Administration (FDA) for use as an inert excipient in pharmaceutical formulations and has, therefore, undergone extensive safety and toxicity studies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. HPβCD is currently under investigation as a potential treatment for Niemann-Pick disease type C (NPC); however, it has not yet received FDA approval for this indication. In the context of NPC, a neurodegenerative disorder characterized by the endolysosomal accumulation of unesterified cholesterol and sphingolipids, HPβCD has been shown to delay the onset of clinical symptoms and reduce accumulation of cholesterol and gangliosides in neuronal cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, HPβCD has proven efficacious in models of atherosclerosis, Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, and Huntington\u0026rsquo;s disease [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHPβCD has also been investigated as an active pharmaceutical ingredient (API) for the prospective treatment of ischemic stroke. We previously demonstrated that repeated administration of HPβCD in young adult and aged mouse models attenuates immune cell infiltration and lipid droplet accumulation in infarcts and improves recovery at transcriptional and functional levels 7 weeks after ischemic stroke [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, we determined that HPβCD induces liver X receptor (LXR)-mediated transcriptional reprogramming in mouse stroke infarcts \u0026ndash; a mechanism that aligns with the HPβCD-induced activation of LXR target genes observed in models of atherosclerotic plaque [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, the efficacy of HPβCD as a treatment for ischemic stroke is dependent on the ability of the therapeutic agent to penetrate ischemic brain tissues, a process influenced by dynamic alterations in BBB integrity and tissue composition over time. The present study aimed to (1) assess the penetration and distribution of FITC-HPβCD within acute and subacute stroke infarcts, which are sites of persisting BBB impairment, and (2) validate the accumulation of FITC-HPβCD in previously identified target organs, including the kidneys, liver, and spleen. Using advanced imaging and analytical techniques, we elucidated the extent of FITC-HPβCD delivery to ischemic brain and peripheral tissues in aged (15-month-old) male mice subjected to stroke induced by distal middle cerebral artery occlusion 24 hours or 1 week prior to subcutaneous administration of FITC-HPβCD. In characterizing the spatiotemporal dynamics of FITC-HPβCD penetration, we seek to provide valuable insights into the potential of HPβCD as a pharmacological intervention for ischemic stroke and inform the development of strategies for enhanced drug delivery to the brain in cerebrovascular diseases.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003e6-deoxy-6-[(5/6)-fluoresceinylthioureido]-(2-hydroxypropyl)-β-cyclodextrin (FITC-HPβCD) was purchased from CarboHyde Zrt. (Identification No. CH-0006). The average degrees of substitution determined by NMR were 1.0 for FITC and 4.5 for (2-hydroxypropyl) groups. FITC-HPβCD underwent extensive dialysis to remove free dye content and 6-deoxy-6-monoamino-HPβCD impurities. FITC-HPβCD, in powder form, was stored at 4\u0026deg;C in a tightly sealed dark glass container to protect from direct light and heat.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Animals\u003c/h2\u003e\u003cp\u003eAged (15-month-old) wild-type male C57BL/6J (Stock No. 000664) mice were purchased from The Jackson Laboratory. Mice were housed in a temperature-controlled suite with a 12-hour light-dark regimen and \u003cem\u003ead libitum\u003c/em\u003e access to food and water. All experimental procedures were conducted in accordance with the animal care standards of the National Institutes of Health and approved by the University of Arizona Institutional Animal Care and Use Committee.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Stroke Surgeries\u003c/h2\u003e\u003cp\u003eStroke was induced in mice using the distal middle cerebral artery occlusion\u0026thinsp;+\u0026thinsp;hypoxia (DH) model. The DH stroke model generates a sizable infarct (24% of the ipsilateral hemisphere centered on the somatosensory cortex), has low variability, and has exceptional long-term survivability. The addition of hypoxia is necessary because C57BL/6 mice that undergo DH stroke without hypoxia have much smaller infarcts. The methodology of the DH stroke model and validation of the controls has been previously published [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To induce stroke, mice were anesthetized by isoflurane inhalation and maintained at 37\u0026deg;C throughout the surgical procedure. For all experiments, mice were injected subcutaneously (s.c.) with a single dose of buprenorphine hydrochloride (0.1 mg/kg) dissolved in sterile saline. Following pre-operative preparation, an incision was made to expose the right temporoparietal skull between the orbit and the ear. Under an operating microscope, a small hole was made with a high-speed microdrill through the outer surface of the semi-translucent skull over the visually identified middle cerebral artery (MCA) at the level of the parietal cerebral artery. Permanent occlusion of the MCA was performed by electrocoagulation with a small vessel cauterizer. Surgical wounds were closed using Surgi-lock 2oc tissue adhesive. Mice were then immediately transferred to a hypoxia chamber containing 9% oxygen and 91% nitrogen for 45 minutes. Extended-release buprenorphine (3.25 mg/kg Ethiqa XR, s.c.) was administered 24 hours after surgery as post-operative analgesia.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. MRI\u003c/h2\u003e\u003cp\u003eInfarct volumes were assessed by MRI 24 hours after stroke using a Bruker BioSpec 70/20 7.0T scanner with ParaVision-360.3.2 software and a 4-channel phase array mouse coil. Mice were placed in a cradle equipped with a stereotaxic frame, an integrated heating system to maintain body temperature at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and a pressure probe to monitor respiration. During MRI acquisition, anesthesia was maintained by inhalation of 1.5-3% isoflurane. High-resolution structural images were acquired using a \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted RARE Bruker pulse sequence with the following parameters: repetition time (\u003cem\u003eTR\u003c/em\u003e)\u0026thinsp;=\u0026thinsp;2500 ms; flip angle\u0026thinsp;=\u0026thinsp;30\u0026deg;; RARE factor\u0026thinsp;=\u0026thinsp;8; matrix size\u0026thinsp;=\u0026thinsp;256 x 256; averages\u0026thinsp;=\u0026thinsp;2; field of view\u0026thinsp;=\u0026thinsp;20 mm x 20 mm; slice thickness\u0026thinsp;=\u0026thinsp;0.8 mm; number of slices\u0026thinsp;=\u0026thinsp;15; acquisition time\u0026thinsp;=\u0026thinsp;2 minutes 40 seconds. Infarcts and hemispheric cross sections were manually delineated on \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MR images using Mango v4.1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. In Vivo and Ex Vivo Fluorescence Imaging\u003c/h2\u003e\u003cp\u003eAt 24 hours or 1 week after stroke, 3.5 mg FITC-HPβCD dissolved in 0.2 mL sterile saline (100 mg/kg FITC-HPβCD, s.c.) was injected into the hip region of each mouse. Mice were anesthetized by isoflurane inhalation and placed in a prone position on the imaging platform of a Spectral Instruments Imaging Lago X. At 0, 5, 10, 15, 20, 25, and 30 minutes after injection of FITC-HPβCD, in vivo fluorescence images were captured (\u0026#120582;\u003csub\u003eex\u003c/sub\u003e: 465 nm; \u0026#120582;\u003csub\u003eem\u003c/sub\u003e: 510 nm). After 30 minutes, perfused organs were dissected for ex vivo imaging. Fluorescence signal strength was quantified using Spectral Instruments Imaging Aura v4.0.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Plasma Collection\u003c/h2\u003e\u003cp\u003eAt 1 and 15 minutes after injection of FITC-HPβCD, a heparinized microhematocrit capillary tube was gently inserted into the retro-orbital sinus, positioned at the medial canthus of the eye. Blood was collected by capillary action into an EDTA-coated microcentrifuge tube. At 30 minutes after injection of FITC-HPβCD, mice were secured in a supine position. The thoracic cavity of each mouse was sterilized with 70% ethanol, and a midline incision was performed to expose the heart. A sterile syringe, equipped with a fine-gauge needle, was inserted into the left ventricle of the heart, and blood was drawn by gentle aspiration. Blood was immediately expelled into an EDTA-coated microcentrifuge tube. Collected blood samples were incubated for 1 hour at 37\u0026deg;C then centrifuged at 4\u0026deg;C for 10 minutes at 5000 x \u003cem\u003eg\u003c/em\u003e to separate blood plasma. Plasma samples were stored at -80\u0026deg;C until further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Tissue Collection\u003c/h2\u003e\u003cp\u003eFollowing exsanguination by intracardiac bleed, mice were perfused with warm 0.9% saline. Organs (brain, heart, liver, spleen, and kidney) were then dissected for ex vivo imaging. Upon completion of ex vivo imaging, organs were prepared for tissue processing. To obtain tissue samples from the heart, liver, spleen, and kidney, smaller pieces of each organ were isolated and crushed with forceps. To obtain tissue samples from the brain, the olfactory bulb and cerebellum were first removed using a brain mold and razor blade. The brain was then divided into contralateral and ipsilateral hemispheres down the longitudinal fissure and stored separately. All tissue samples were flash frozen in liquid nitrogen and stored at -80\u0026deg;C until further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Tissue Processing\u003c/h2\u003e\u003cp\u003eTissue samples were processed prior to total protein quantification in batches of 10\u0026ndash;15 samples. Samples were placed on ice and allowed to thaw for 5 minutes. 500 \u0026micro;L of radioimmunoprecipitation assay (RIPA) buffer was added to heart, liver, spleen, and kidney samples, and 1 mL of RIPA buffer was added to brain samples. Samples were sonicated then centrifuged at 4\u0026deg;C for 15 minutes at 13,000 x \u003cem\u003eg\u003c/em\u003e. Supernatant was collected from each sample and placed in a 1.5 mL Eppendorf tube. Total protein quantification was performed using a Millipore Direct Detect Spectrometer according to manufacturer instructions. FITC-HPβCD content of each sample was normalized to tissue protein content.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Microplate Reader Quantification of FITC-HPβCD\u003c/h2\u003e\u003cp\u003eAliquots of plasma and tissue samples were placed into black 96-well plates, and fluorescence intensities were measured using a BioTek Cytation 3 Microplate Reader according to manufacturer instructions (\u0026#120582;\u003csub\u003eex\u003c/sub\u003e: 492 nm; \u0026#120582;\u003csub\u003eem\u003c/sub\u003e: 520 nm). Each standard, experimental plasma sample, and experimental tissue sample was measured in duplicate. The lower limit of detection for FITC-HPβCD in mouse plasma and tissue samples was 0.01 \u0026micro;g/mL. Fluorescence intensities were quantified using BioTek Gen5 v2.06.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism v10.2.3. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Differences were considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Full statistical results, including the statistical tests, number of observations, test statistics, exact probabilities, and degrees of freedom, are reported in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1. In vivo imaging of FITC-HPβCD in aged mice 24 hours after stroke\u003c/h2\u003e\u003cp\u003eTo assess the accumulation and distribution of FITC-HPβCD in the acute and subacute phases after stroke, aged (15-month-old) male mice were subjected to DH stroke. At 24 hours after stroke, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MRI was performed to confirm infarct generation. At 24 hours or 1 week after stroke, mice were injected with FITC-HPβCD (100 mg/kg, s.c.) and immediately placed in a Spectral Instruments Imaging Lago X for in vivo imaging. At 0, 5, 10, 15, 20, 25, and 30 minutes after injection of FITC-HPβCD, serial images of mice were captured. At 1, 15, and 30 minutes after injection of FITC-HPβCD, plasma was collected for fluorescence quantification on a microplate reader. Upon completion of in vivo imaging and subsequent plasma collection at 30 minutes after injection of FITC-HPβCD, mice were perfused with warm saline, and organs (i.e., heart, brain, liver, spleen, and kidney) were dissected for ex vivo imaging and subsequent fluorescence quantification on a microplate reader \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt 24 hours after stroke, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MRI was performed to confirm infarct generation and ensure proper stratification of mice across experimental groups. The quantification of \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MRI scans verified that acute infarct volume did not differ significantly between groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Upon completion of \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e-weighted MRI, mice were injected with FITC-HPβCD (100 mg/kg, s.c.) and immediately placed in a Spectral Instruments Imaging Lago X for in vivo imaging. The quantification of serial in vivo images revealed increasing fluorescence intensities over 30 minutes in mice injected with FITC-HPβCD. Notably, FITC-HPβCD disseminated comparably throughout the bodies of both na\u0026iuml;ve mice and those subjected to DH stroke 24 hours prior. Additionally, minimal fluorescence was observed in uninjected na\u0026iuml;ve mice and those subjected to DH stroke 24 hours prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Ex vivo imaging of FITC-HPβCD in organs from aged mice 24 hours after stroke\u003c/h2\u003e\u003cp\u003eAt 30 minutes after injection of FITC-HPβCD, organs (i.e., heart, brain, liver, spleen, and kidney) were dissected for ex vivo imaging. The quantification of ex vivo images revealed that FITC-HPβCD can permeate the brain 24 hours after DH stroke; FITC-HPβCD localized primarily to the ipsilateral (i.e., infarcted) hemisphere \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Importantly, FITC-HPβCD was processed similarly in the livers and kidneys of both na\u0026iuml;ve mice and those subjected to DH stroke 24 hours prior; however, FITC-HPβCD accumulated to a greater extent in the brains of mice subjected to DH stroke compared to brains of na\u0026iuml;ve mice. Additionally, minimal fluorescence was detected in the spleens and hearts of uninjected and injected mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3. FITC-HPβCD concentrations in organs from aged mice 24 hours after stroke\u003c/h2\u003e\u003cp\u003eUpon completion of ex vivo imaging, organs were processed for subsequent fluorescence quantification on a microplate reader. FITC-HPβCD was predominantly localized to the kidneys 30 minutes after injection in na\u0026iuml;ve mice and those subjected to DH stroke 24 hours prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. However, FITC-HPβCD was also detected in the livers and hearts of na\u0026iuml;ve mice and those subjected to DH stroke 24 hours prior. Interestingly, FITC-HPβCD accumulated in the spleens of mice subjected to DH stroke 24 hours prior but did not accumulate significantly in the spleens of na\u0026iuml;ve mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Additionally, FITC-HPβCD was concentrated in the ipsilateral (i.e., infarcted) hemispheres compared to the corresponding contralateral hemispheres of brains dissected from mice subjected to DH stroke 24 hours prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. FITC-HPβCD also temporally accumulated in the plasma of na\u0026iuml;ve mice and mice subjected to DH stroke 24 hours prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4. In vivo imaging of FITC-HPβCD in aged mice 1 week after stroke\u003c/h2\u003e\u003cp\u003eTo assess the accumulation and distribution of FITC-HPβCD in the subacute phase after stroke, aged (15-month-old) male mice were subjected to DH stroke 1 week prior to injection of FITC-HPβCD. The quantification of serial in vivo images revealed increasing fluorescence intensities over 30 minutes in mice injected with FITC-HPβCD. Analogous to the distribution of FITC-HPβCD in the acute phase after stroke, FITC-HPβCD disseminated comparably throughout the bodies of both na\u0026iuml;ve mice and those subjected to DH stroke 1 week prior. Additionally, minimal fluorescence was observed in uninjected na\u0026iuml;ve mice and those subjected to DH stroke 1 week prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Ex vivo imaging of FITC-HPβCD in organs from aged mice 1 week after stroke\u003c/h2\u003e\u003cp\u003eAt 30 minutes after injection of FITC-HPβCD, organs (i.e., heart, brain, liver, spleen, and kidney) were dissected for ex vivo imaging. The quantification of ex vivo images revealed that FITC-HPβCD can permeate the brain 1 week after DH stroke; FITC-HPβCD localized primarily to the ipsilateral (i.e., infarcted) hemisphere. Notably, subacute stroke infarcts in uninjected mice exhibited green-channel autofluorescence (GCAF), consistent with prior observations in mouse models of ischemic stroke [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. GCAF can be attributed to the intracellular accumulation of lipofuscin, an autofluorescent lipid-rich polymeric entity, caused by oxidative stress [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. FITC-HPβCD accumulated similarly in the livers, kidneys, and hearts of both na\u0026iuml;ve mice and those subjected to DH stroke 1 week prior; however, FITC-HPβCD accumulated to a greater extent in the brains of mice subjected to DH stroke compared to brains of na\u0026iuml;ve mice. Interestingly, FITC-HPβCD was detected in the spleens of mice subjected to DH stroke 1 week prior when compared to uninjected na\u0026iuml;ve mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6. FITC-HPβCD concentrations in organs from aged mice 1 week after stroke\u003c/h2\u003e\u003cp\u003eUpon completion of ex vivo imaging, organs were processed for subsequent fluorescence quantification on a microplate reader. FITC-HPβCD was predominantly localized to the kidneys 30 minutes after injection in na\u0026iuml;ve mice and those subjected to DH stroke 1 week prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. However, FITC-HPβCD was also detected in the livers, spleens, and hearts of na\u0026iuml;ve mice and those subjected to DH stroke 1 week prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Additionally, FITC-HPβCD was concentrated in the ipsilateral (i.e., infarcted) hemispheres compared to the corresponding contralateral hemispheres of brains dissected from mice subjected to DH stroke 1 week prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. FITC-HPβCD also temporally accumulated in the plasma of na\u0026iuml;ve mice and mice subjected to DH stroke 1 week prior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHPβCD, a chemically modified derivative of β-cyclodextrin, has emerged as a versatile molecule with numerous applications in the pharmaceutical sector. Initially patented as an excipient nearly forty years ago, HPβCD is commonly utilized to enhance the aqueous solubility, stability, and bioavailability of steroids, antivirals, and chemotherapies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. HPβCD has also more recently been investigated as an API. These investigations were prompted by its widely approved safety profile and capacity to interact with biomolecules, such as cholesterol. HPβCD is included in both the European Pharmacopoeia (PhEur) and United States Pharmacopoeia (USP) and received an orphan drug designation for the treatment of NPC, although it has not yet received FDA approval for this indication. Currently, research is ongoing to evaluate the therapeutic efficacy of HPβCD in related diseases, including atherosclerosis, Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, and ischemic stroke, all of which exhibit pathological accumulation of aggregation-prone peptides, proteins, or lipids in various tissues [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eExtensive investigations into the toxicity, metabolism, and pharmacokinetics of HPβCD in humans and animals have consistently demonstrated that it is well tolerated across species [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These studies have determined that parenteral (e.g., intravenous and subcutaneous) administration of HPβCD is characterized by rapid distribution within the extracellular fluid compartment due to its hydrophilicity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. HPβCD is not significantly metabolized by the liver or other tissues and is primarily eliminated intact in the urine via renal excretion. In humans, the plasma half-life after intravenous administration is relatively short, ranging from 1 to 2 hours [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, HPβCD does not readily cross lipid membranes, including the BBB, unless administered intrathecally [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, we aimed to (1) assess the penetration and distribution of FITC-HPβCD within acute and subacute stroke infarcts, which are sites of persisting BBB impairment, and (2) validate the accumulation of FITC-HPβCD in previously identified target organs, including the kidneys, liver, and spleen.\u003c/p\u003e\u003cp\u003eGould and Scott (2005) previously reviewed several toxicology studies indicating that intravenous administration of HPβCD affected main target organs, including the kidneys, liver, lungs, and spleen [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In accordance with published human and animal studies, we determined that FITC-HPβCD was predominantly localized to the kidneys 30 minutes after subcutaneous administration in both na\u0026iuml;ve mice and those subjected to DH stroke 24 hours or 1 week prior, further substantiating renal excretion as the primary route of elimination. Importantly, V\u0026aacute;radi et al. reported a comparable accumulation of FITC-HPβCD in the kidneys of male BALB/c mice 60 minutes after intravenous administration [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition to the kidneys, FITC-HPβCD was detected in the livers and, to a lesser extent, in the hearts and spleens of both na\u0026iuml;ve mice and those subjected to DH stroke 24 hours or 1 week prior. It is plausible that the modest concentrations of FITC-HPβCD observed in the hearts and spleens may be attributed to its endocytic uptake by cardiac and splenic endothelial cells, a phenomenon previously described in human umbilical vein endothelial cells (HUVECs) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These results indicate that HPβCD is distributed and excreted comparably after subcutaneous and intravenous administration and provide valuable insights into the pharmacokinetics and tissue-specific accumulation of HPβCD during the acute and subacute phases of ischemic stroke recovery.\u003c/p\u003e\u003cp\u003eFITC-HPβCD also selectively accumulated in the ipsilateral (i.e., infarcted) hemispheres compared to the contralateral hemispheres of brains at 24 hours or 1 week after DH stroke. The preferential accumulation of FITC-HPβCD in the ipsilateral (i.e., infarcted) hemisphere reflects the increased permeability of the BBB in the affected region(s), a recognized hallmark of ischemic stroke [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These results are particularly relevant to the potential application of HPβCD in facilitating the clearance of cholesterol and other lipids implicated in ischemic stroke pathology, which our previous studies indicate drive secondary neurodegeneration through inflammatory mechanisms [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, there are several methodological limitations that must be acknowledged. Firstly, these imaging and analytical analyses were conducted exclusively in aged (15-month-old) male C57BL/6J mice. We recognize the necessity of repeating these analyses, particularly the in vivo imaging, in male and female BALB/c mice due to their lighter coat color, which would allow for improved visualization of FITC-HPβCD. However, V\u0026aacute;radi et al. reported that FITC-HPβCD produced a potent fluorescent signal in the highly perfused skin capillaries of BALB/c mice, which concealed fluorescent signals from specific internal organs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Secondly, we opted to administer a single dose of 100 mg/kg FITC-HPβCD via subcutaneous injection 24 hours or 1 week after DH stroke. Future studies should incorporate the following: (1) therapeutic concentrations of FITC-HPβCD ranging from 100 mg/kg to 4000 mg/kg, (2) intravenous or intraperitoneal routes of administration, (3) models of ischemic stroke involving reperfusion (e.g., intraluminal filament MCA occlusion), and (4) additional end points ranging from 2 weeks to 8 weeks after ischemic stroke.\u003c/p\u003e\u003cp\u003eIn conclusion, the present study bolsters the potential of HPβCD as an API for the treatment of ischemic stroke. The observed pharmacokinetic profile, characterized by renal excretion as the primary route of elimination and selective accumulation in infarcted brain regions with compromised BBB integrity, underscores its capacity to facilitate the clearance of cholesterol and other lipid byproducts resulting from myelin and cellular membrane degradation after ischemic stroke. Combined with its well-established safety profile, these results provide a strong rationale for further investigation into the therapeutic applications of HPβCD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics Approval and Consent to Participate\u003c/em\u003e\u003c/strong\u003e:All experimental procedures were conducted in accordance with the animal care standards of the National Institutes of Health and approved by the University of Arizona Institutional Animal Care and Use Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for Publication\u003c/em\u003e\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of Data and Materials\u003c/em\u003e\u003c/strong\u003e: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e: This research was funded by the National Institute of Neurological Disorders and Stroke RF1NS131110 (K.D.), National Institute on Aging R01AG063808 (K.D.), United States Department of Veterans Affairs I01RX003224 (R.S.), National Institute on Aging T32AG058503-01A1 (D.B.), and the Fondation Leducq Transatlantic Network of Excellence Stroke-IMPaCT (K.D.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026rsquo; Contributions\u003c/em\u003e\u003c/strong\u003e: Conceptualization, D.B. and K.D.; Methodology, D.B., J.F., S.W. and K.D.; Formal Analysis, D.B., E.L., J.F. and S.W.; Investigation, D.B., E.L., J.F. and S.W.; Resources, K.D.; Writing \u0026ndash; Original Draft, D.B. and E.L.; Writing \u0026ndash; Review \u0026amp; Editing, R.S. and K.D.; Visualization, D.B., E.L. and K.D.; Supervision, K.D.; Project Administration, D.B. and K.D.; Funding Acquisition, R.S. and K.D.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e: We thank Dr. Milo Malanga at CarboHyde Zrt. for his efforts in the synthesis and characterization of FITC-HP\u0026beta;CD.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMartin SS et al. 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Neurobiol Dis. 2018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.nbd.2018.01.007\u003c/span\u003e\u003cspan address=\"10.1016/j.nbd.2018.01.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChung AG et al. \u0026lsquo;Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis and mediates secondary neurodegeneration in an osteopontin-dependent mechanism\u0026rsquo;, \u003cem\u003eeNeuro\u003c/em\u003e, vol. 5, no. 5, 2018, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/ENEURO.0076-18.2018\u003c/span\u003e\u003cspan address=\"10.1523/ENEURO.0076-18.2018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Stroke, Brain, 2-Hydroxypropyl-β-Cyclodextrin, Cyclodextrin, Fluorescence","lastPublishedDoi":"10.21203/rs.3.rs-7521331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7521331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Ischemic stroke is a leading cause of mortality and long-term disability worldwide, with limited pharmacological interventions available. 2-hydroxypropyl-β-cyclodextrin (HPβCD), a cyclic oligosaccharide approved for the treatment of Niemann-Pick disease type C, has demonstrated therapeutic potential in preclinical models of ischemic stroke by attenuating immune cell and lipid droplet accumulation in infarcts. However, the ability of HPβCD to penetrate ischemic brain tissues remains a critical determinant of its efficacy. The present study aimed to (1) assess the penetration and distribution of FITC-HPβCD within acute and subacute infarcts, which are sites of persisting blood-brain barrier (BBB) impairment, and (2) validate the accumulation of FITC-HPβCD in previously identified target organs, including the kidneys, liver, and spleen, using an aged (15-month-old) male mouse model of ischemic stroke induced by distal middle cerebral artery occlusion. We determined that FITC-HPβCD exhibits widespread systemic dissemination within 30 minutes after subcutaneous administration and is primarily eliminated via renal excretion. Notably, FITC-HPβCD selectively accumulated in the ipsilateral (i.e., infarcted) hemisphere 24 hours and 1 week after ischemic stroke, indicating that ischemia enhances the penetration of FITC-HPβCD into the brain. These results provide valuable insights into the therapeutic potential of HPβCD as a treatment for ischemic stroke and inform strategies for optimizing drug delivery to the brain in cerebrovascular diseases.","manuscriptTitle":"2-Hydroxypropyl-β-Cyclodextrin Accesses Acute and Subacute Infarcts in a Mouse Model of Ischemic Stroke","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 17:49:36","doi":"10.21203/rs.3.rs-7521331/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-04T03:25:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T15:02:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-17T16:18:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153089400326146221759104898273112281571","date":"2025-09-11T17:00:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300200706558473121602350464173601373710","date":"2025-09-09T21:25:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272773688193306635549456915736137771593","date":"2025-09-09T21:21:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-07T21:18:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T19:46:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-04T18:14:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fluids and Barriers of the CNS","date":"2025-09-02T22:51:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2b5dbd7-f87f-44b6-911f-d4eb90feaf8d","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:08:06+00:00","versionOfRecord":{"articleIdentity":"rs-7521331","link":"https://doi.org/10.1186/s12987-026-00767-9","journal":{"identity":"fluids-and-barriers-of-the-cns","isVorOnly":false,"title":"Fluids and Barriers of the CNS"},"publishedOn":"2026-01-28 15:58:19","publishedOnDateReadable":"January 28th, 2026"},"versionCreatedAt":"2025-09-12 17:49:36","video":"","vorDoi":"10.1186/s12987-026-00767-9","vorDoiUrl":"https://doi.org/10.1186/s12987-026-00767-9","workflowStages":[]},"version":"v1","identity":"rs-7521331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7521331","identity":"rs-7521331","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}