Hypoglycemia rather than vascular dysfunction causes early mortality in diabeto-septic mice

Hypoglycemia rather than vascular dysfunction causes early mortality in diabeto-septic mice | 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 Hypoglycemia rather than vascular dysfunction causes early mortality in diabeto-septic mice Manju Gari, T Jagadeesh, Soumen Choudhury, Amit Shukla, Neeraj K Gangwar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3857212/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 Sepsis is often complicated with pre-existing diabetes and diabetic patients are prone to infection. However, the impact of sepsis in pre-existing diabetes especially on cardio-vascular system is largely unknown. Sepsis was induced by caecal ligation and puncture while intra-peritoneal injection of streptozotocin (@ 65 mg/kg b.wt. for 5 consecutive days) was used to induce diabetes in mice. Isometric tension and mRNA expression of α 1D -adrenoceptor in aorta was determined by organ bath and qRT-PCR, respectively. Blood glucose levels and bacterial load in blood and peritoneal lavage (PL) were estimated. Histopathological examination of pancreas, lungs, liver, kidney and spleen was also done. Induction of sepsis in the mice with pre-existing diabetes caused early mortality despite being lower bacterial load in blood and PL in comparison to sepsis alone. Interestingly, NA-induced contraction as well as receptor-independent high K + -induced contraction (though significantly ( p < 0.05) reduced in sepsis), were similar in diabeto-septic and SO groups. Accordingly, aortic mRNA expression of α 1D -adrenoceptor was also unaltered in diabeto-septic group unlike to that of sepsis where α 1D mRNA expression was significantly down-regulated. ACh-induced vasorelaxation was also unaffected in these animals. However, marked hypoglycemia before death with enhanced infiltration of inflammatory cells in lungs, liver, kidney and spleen was observed. In diabeto-septic animals, hypoglycaemia rather than vascular dysfunction was responsible for early mortality. Further, the increased infiltration of inflammatory cell in different tissues reduced the bacterial load and is responsible, at least in part, for reduction in blood glucose level leading to hypoglycemic shock. sepsis diabetes hypoglycemia vascular dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Sepsis is a multifactorial disease with uncontrolled systemic production of inflammatory mediators (‘cytokine storm’) that leads to systemic inflammatory response syndrome (SIRS) following systemic microbial infection [ 1 ]. Sepsis ranks tenth leading cause of deaths in the United States with mortality rates varying between 30 and 70% among ICU patients [ 2 – 4 ]. Sepsis is often complicated by other co-morbidity conditions especially cardiovascular disorders and diabetes [ 5 ]. Local and systemic (sepsis) infections are frequently encountered in patients with diabetes [ 6 ] and regardless of the type, diabetic patients have higher risks of infection [ 7 , 8 ]. As per the available information, approximately 22% mortalities in diabetic human patients are associated with infections [ 9 – 10 ]. In experimental animals too, diabetic mice have been reported to be highly susceptible to polymicrobial sepsis [ 11 – 15 ]. However, most of the preclinical sepsis studies in laboratory animals have been typically conducted in healthy animals without considering the clinical relevance or impact of co-morbidities due to diabetes and sepsis. Hyperglycemia, abnormalities in renin-angiotensin axis, enhanced vascular smooth muscle contraction, endothelial dysfunctions, and nephropathy are some of the reasons for the development of hypertension in diabetes [ 16 ]. On the other hand, circulatory failure in septic patients is mainly due to hypotension leading to hypo-perfusion of majority of the body organs. Therefore, in a pathological situation, where pre-existing hypertension (like in diabetes) precedes the hypotensive state (like in sepsis), a more realistic and complex clinical condition is likely when diabetes and sepsis co-exist, and thus, vascular reactivity is likely to be affected in a different way. Numerous studies have documented an increase in the sensitivity of vascular smooth muscles to noradrenaline (NA) in arteries from diabetic animals [ 17 – 21 ] while vascular hyporeactivity to vasoconstrictors including NA is considered to be the underlying cause of death in sepsis [ 22 , 23 ]. Thus, we aimed to study the effect of sepsis on vascular dysfunction in pre-existing diabetes mice model in an endeavour to see whether the death of diabeto-sepsis animals occurs because of vasoplegia or some other reason. Materials and methods Experimental animals Adult male Swiss albino mice (26–28 g) were procured from Disease Free Small Animal House, College of Veterinary and Animal Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, and kept in polypropylene cages in the Departmental Laboratory Animal House under 12–12 h dark-light cycle. Experimental animals had free access to pellet feed (Ashirwad Industries, Mohali, Punjab) and drinking water. An acclimatization period of 10 days was allowed before starting the actual study. The study was undertaken after obtaining approval from the Institutional Animal Ethics Committee (IAEC) of the University (Approval No.: IAEC/17/12, Dated 26-07-2017). Drugs and chemicals Streptozotocin (STZ), acetylcholine (ACh), nor-adrenaline (NA), and phenylephrine (PE) were procured from Sigma-Aldrich (St. Louis, Mo, USA). All other used chemicals/reagents were of analytical grade. Noradrenaline (NA) was dissolved in 0.1 N hydrochloric acid plus 0.01% ascorbic acid. Streptozotocin was dissolved in 0.1 M citrate buffer solution. All other chemicals were dissolved in distilled water. Induction of diabetes Type-1 diabetes in mice was induced by administering five sequential daily intra-peritoneal injections of freshly prepared streptozotocin solution (STZ @ 65 mg/kg b.w.) as described earlier [ 24 ]. Streptozotocin solution was prepared immediately before use and injected within 5 min of dissolution as STZ degrades within 15–20 min after dissolving in citrate buffer. Body weights of animals and blood glucose levels were monitored at the weekly interval to assess the progression of diabetes. Animals showing > 300 mg/dl blood glucose levels were considered to be diabetic. 10% sucrose solution in water was provided to mice by oral route to avoid hypoglycemia. Induction of sepsis Sepsis was induced by the caecal ligation and puncture method (CLP) in mice as described earlier [ 25 , 26 ]. Briefly, following overnight fasting, mice were anesthetized with xylazine (10 µg/g b.wt, i.p ) and ketamine (80 µg/g b.wt, i.p. ). A midline incision (2cm long) was made in the abdominal region to expose the cecum and thereafter the cecum was ligated with 2 − 0 silk distal to the ileocecal valve to avoid intestinal obstruction. The cecum was then punctured twice with a 21G needle and placed back into the abdominal cavity. Then the abdominal incision was closed in layers. To prevent dehydration, isotonic sodium chloride solution (1 mL/mouse) was subcutaneously injected to all the mice. Sham-operated (SO) mice were subjected to the same surgical procedure except CLP, and served as control. After induction of sepsis or only surgical procedure in mice of SO group, animals were closely observed for up to 72 h for development of sepsis based on lethargy, induction of conjunctivitis, absence of grooming behavior, ruffled fur, and reduced feed and water intake or any other apparent change in behavior of animals including mortality. Induction of diabeto-sepsis In a separate group of mice, first diabetes was induced by administering STZ as mentioned above and after ensuring the development of diabetes (eight weeks after administration of the first dose of STZ), the animals were subjected to caecal ligation and puncture to induce sepsis. Survival times: Animals of all the four groups were closely observed for any signs of discomfort or survival for up to 72 hours. The survival curves and mean survival times for mice of all the groups were plotted by Kaplan-Meier survival curve method and analyzed using log-rank test. Determination of total bacterial load Total bacterial load in blood (systemic infection) and peritoneal fluid (source of infection) were determined as mentioned earlier [ 23 ] for assessment of sepsis. Briefly, blood samples were collected by cardiac puncture under xylazine-ketamine anesthesia. For the collection of the peritoneal lavage (PL), 2 ml sterile phosphate buffer saline (PBS) was injected into the peritoneal cavity at the time of sacrifice of animal and PL was aseptically collected after giving an incision in the abdominal region and before collecting the organs of interest for other studies. The samples (blood and PL) were then serially diluted (1:10) in sterile phosphate buffer saline (PBS) and 100µl of each dilution was plated on Luria-Bertani agar plates and incubated at 37 ° C for 18 h. Each dilution was plated in duplicate and bacterial loads were expressed as log CFU/ml. Collection of tissues for functional and molecular studies: Mice of the diabetes, sepsis, diabeto-sepsis and SO groups were sacrificed by bleeding from vena cava under xylazine-ketamine anaesthesia after 18 h of the CLP or SO procedure. Thorax and abdomen were cut open and lungs and heart were taken out en-bloc along with the thoracic aorta and immediately placed in ice-cold (4 ° C) Modified Krebs-Henseleit solution (MKHS- 118.0 NaCl, 4.7 KCl, 2.5 CaCl 2 ·2H 2 O, 1.2 MgSO 4 ·7H 2 O, 1.2 KH 2 PO 4 , 11.9 NaHCO 3, and 11.1 glucose mmol/L). Thoracic aorta was cleaned off the adhering connective tissues under a dissection stereo- microscope (Motic, China) and aortic rings of 3–4 mm length were prepared without damaging the endothelium. To study the endothelium-independent response of aortic rings, endothelial denudation was undertaken by passing horse tail hair through the arterial rings. Measurement of isometric tension Aortic rings from the mice of different treatment groups were mounted between two “L” shaped hooks made from 37 G stainless steel wire and mounted under a resting tension of 1.0 g in thermostatically controlled (37 ± 1 ° C) organ bath (Radnoti, USA) of 10 ml capacity containing modified Krebs–Henseleit solution (MKHS) continuously bubbled with medical gas (74% N 2 + 21% O 2 + 5% CO 2 ). Isometric tension was measured using the high-sensitivity isometric force transducer and recorded in a PC using LabChart V6.1.3 Pro software programme (Powerlab, AD Instruments, Australia). Before starting any experimental protocol, tissue viability was assessed by recording the aortic contraction to a high K + (80 mM) depolarizing solution. Nor-adrenaline is commonly used as a vasopressor agent in septic patients and it also has a role in hypertension in diabetic patients. Therefore, cumulative concentrations response to NA (0.1 nM – 10 µM) at an increment of 0.5 log concentration unit was studied in the arterial rings of the mice from different groups. In a separate set of experiment, to assess the alterations in endothelium-dependent relaxant responses of the aortic rings, concentration-dependent responses to acetylcholine (ACh; 1 nM–10 µM) was also recorded in phenylephrine (PE)-pre-contracted aortic rings from the mice from different groups. Before eliciting the relaxant response to ACh in the aortic rings from mice of different groups, the matching mean pre-contractile tensions (0.35 ± 0.02 g) were generated in all the tissues using phenylephrine. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) Thoracic aorta was collected in 0.1% diethyl pyrocarbonate (DEPC) treated autoclaved phosphate buffer saline (PBS), and after removing the adjacent fat and connective tissues, samples were quickly snap-frozen in liquid nitrogen and stored in RNA later at − 80°C until further use. Total RNA was isolated using commercially available kit (Ambion, Thermo scientific) by following the manufacturer’s protocol. Samples were then treated with RNase-free DNase and DNase was subsequently inactivated by heating at 56°C for 10 min and immediately chilled at 4°C. The purity of RNA was checked by biophotometer (Eppendorf, USA). cDNA synthesis (from 150 ng total RNA) was carried out using Revertaid® First strand cDNA synthesis kit (Thermo Scientific, USA) using Moloney murine leukemia viral reverse transcriptase enzyme by following the manufacturer’s instructions. qRT-PCR reactions were performed in duplicate using SYBR Green chemistry (PowerUp TM SYBR™ Green master mix [2X]; ThermoFischer Scientific, USA) in QuantStudio 3, Applied Biosystems). Each reaction was consisted of SYBR Green master mix (5 µl), gene-specific forward and reverse primers (0.5 µL each of 10 pmol/µL stock) and cDNA (1 µl) in a total volume of 10 µL. The real-time PCR reaction was started with initial incubation at 95°C for 10 min followed by 42 cycles of amplification with denaturation at 95°C for 1 min, annealing at (temperature as mentioned below) for 1 min and extension at 72°C for 1 min each. The optimum annealing temperature determined by PCR for α 1D -adrenoceptor specific primer sets (F5′-GCCTCTGAGGTGGTTCTGAG-3′, R 5′-GGACGAAGAAAAAGGGGAAC-3′; 208 bp) was 57°C and for GAPDH (the reference gene- F 5′-AACTTTGGCATTGTGGAAGG-3′, R 5′ ACACATTGGGGGTAGGAACA-3′; 223 base pairs) was 58°C. To assess the specificity of the amplified product, dissociation curve was generated at temperature of 60°C through 95°C. The results were expressed as threshold cycle values (C T ). Histopathological studies: Pancreas, liver, lungs, kidney, and spleen were collected from animals of all the four groups (SO, diabetes, sepsis and diabeto-sepsis) in 10% buffered formal saline and kept for 72 h for fixation. Tissue sections of approximately 4–5 µm thickness were prepared and stained using Hematoxylin and Eosin (H & E) stain and examined under light microscope (Nikon, Japan) to determine the pathological lesions in different organs of the mice of different groups. Special staining of the pancreatic sections was undertaken using Gomori’s Chromium-hematoxylin-phloxine stain for determining the extent of destruction of beta cells of the pancreas [ 27 ]. The scoring of histopathological lesions was done as described earlier [ 28 ]. Statistical analysis Data are presented as mean ± SEM and ‘n’ refers to the number of animals used in different experimental protocols. The overall difference in survival rate was determined by the Kaplan–Meier test followed by the log-rank test. For measurement of the bacterial colony-forming units (CFU), all data points are presented. Groups were compared by using the nonparametric Kruskal–Wallis test followed by a post-hoc Dunn's test. The E max (the maximal response) and the EC 50 (the concentration producing 50% of the maximal response) values were determined by non-linear regression analysis using GraphPad Prism V.4.00 (San Diego, California). Potency is defined as the pD 2 value which is the -log of EC 50 value. Concentration-dependent agonist response data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test by using GraphPad Prism. To study the relative change in gene expression, the 2 −∆∆C T method was used as described earlier [ 29 ]. The formula used to calculate the “fold change in gene expression” was = 2 −∆∆C T ,” [where ∆∆C T = (C T,target gene - C T,GAPDH ) treatment - (C T,target gene -C T,GAPDH ) control]. The gene-specific amplification was corrected for the difference in input of RNA by taking house-keeping gene GAPDH to account. For diabetes, sepsis and diabeto-sepsis groups, evaluation of 2 −∆∆C T indicates the fold change in gene expressions relative to SO control (SO control = 1). The results were analyzed in comparison with the C T (minimum threshold of amplification) value of the target gene and the reference gene (GAPDH). Difference in values was considered statistically significant at p < 0.05. Results A) Assessment of hyperglycemia and associated vascular dysfunctions in diabetic mice i) Effect on blood glucose levels and body weight gain: Blood glucose levels were measured at weekly interval and the data are summarized in Table S-1 (Supplementary file). Mean blood glucose level of > 300 mg/dl (hyperglycemia) was observed in animals of the diabetes group from 3 rd week onwards following STZ administration and this hyperglycemic level was maintained up to 8 th week of observation period. Compared to the body weights of animals of both the groups (healthy control and diabetes) on day 0, body weight gain after eight weeks in mice of the diabetes group was significantly lower than in the animals of healthy control group (Supplementary Table S2). ii) Effect on high K + (80 mM)-depolarizing solution-induced contraction: Given that vascular dysfunction(s) are late manifestation of diabetes, we compared the vascular reactivity to different spasmogens and relaxant agents in aortic rings of the diabetic mice sacrificed after 5 th and 8 th weeks of STZ administration. As shown in Fig. 1A the maximal contraction induced by high K + (80 mM)-depolarizing solution (KDS) was found to be significantly ( p <0.05) higher (0.65 ± 0.02 g, n=10) after 8 th week of STZ administration as compared to that observed in healthy control group (0.50 ± 0.02 g; n=10). However, High K + -induced contraction was found to be statistically similar in the diabetic mice after 5 th week of STZ administration (0.56 ± 0.03 g, n=10) as compared to that observed in healthy control group (0.50 ± 0.02 g; n=10). iii) Effect on noradrenaline (NA)-induced contraction: Noradrenaline (NA) produced concentration-dependent (0.1 nM – 10 µM) contraction in aortic rings of the mice of healthy control group. As shown in Fig.1B, the cumulative concentration-response curve of NA in diabetes group after 8 th week of STZ administration was significantly ( p <0.05) shifted towards left with increase in the maximal contraction as compared to healthy control (E max : 0.75 ± 0.04 g vs. 0.51 ± 0.04 g, n=8). However, no significant change was observed in the maximal contraction of NA in 5 th week diabetic group (0.53 ± 0.03 g; n=8) in comparison to healthy control. B) Assessment of vascular dysfunctions in septic (CLP) mice i) Effect on survival time: Fig.2A shows the survival curves and mean survival times of the mice of sepsis (CLP) and sham-operated (SO) groups. All the mice of SO group (n=15) survived during the observation period of 72 h while the mean survival time in mice of the sepsis group (18.15 ± 0.18 h; n=11) was found to be significantly ( p <0.001) lower. ii) Effect on bacterial load: No bacterial colonies were observed either in blood or in the peritoneal lavage of the mice from SO group. However, comparatively higher total bacterial load was observed in the blood (6.58 ± 0.26 Log CFU/ml, n=6) and peritoneal lavage (10.44 ± 0.13 Log CFU/ml, n=6) of the mice from septic group in comparison to SO group. iii) Effect on high K + (80 mM)-depolarizing solution-induced contraction: As illustrated in Fig. 2B, sepsis significantly ( p <0.05) attenuated the maximum contraction elicited by 80 mM KDS (0.27 ± 0.01 g; n=10) in the aortic rings after 18h of CLP as compared to that observed in SO group (0.51 ± 0.01 g, n=10). iv) Effect on NA-induced contraction: As shown in Fig. 2C, in comparison to the aortic response in mice of SO group, sepsis significantly ( p <0.05) reduced the maximal contractile response to NA in the aortic rings isolated 18h post-CLP (E max : 0.51 ± 0.02 g vs. 0.24 ± 0.02 g; n=8) but without any change in the potency (pD 2 : 7.45 ± 0.05 vs. 7.43 ± 0.07, n=8). C) Effect of sepsis on vascular function in pre-existing diabetic mice i) Effect on survival time: As shown in the Fig.3A, septic mice with pre-existing diabetes (diabeto-septic group) showed early mortality (mean survival time: 13.23 ± 0.36 h; n=9) as compared to the septic mice (18.15 ± 0.18 h; n=11). Thus, in further study to compare the vascular reactivity of the mice between sepsis and diabeto-sepsis groups, mice were sacrificed 13 h post-CLP. ii) Effect on blood glucose level: Time-dependent progression of blood glucose level after induction of sepsis was measured and illustrated in Fig.3B. In septic mice, blood glucose level exhibited three different phases, namely- early hyperglycaemic phase (2-10 h post CLP), followed by euglycaemic phase (12-16 h post CLP) and late hypoglycaemic phase (18-20 h post CLP) whereas the septic mice with pre-existing diabetes showed only early hyperglycaemia (2-10 h post CLP) followed by sharp hypoglycaemic phase (12-14 h post CLP). Diabetic mice showed consistently higher blood glucose level. iii) Effect on bacterial load: As shown in Fig. 3C&D, in the septic mice with pre-existing diabetes, total bacterial count was comparatively lower in blood (5.26 ± 0.19 Log CFU/ml, n=6) and peritoneal lavage (9.15 ± 0.27 Log CFU/ml, n=6) as compared to the respective counts in the septic mice (6.58 ± 0.26 Log CFU/ml and 10.44 ± 0.13 Log CFU/ml, n=6, respectively). However, the live bacterial counts in blood and peritoneal lavage in both septic and diabeto-septic mice were found to be significantly higher in comparison to SO and diabetic mice. No bacterial count was detected in blood and peritoneal lavage from SO and diabetic mice. iv) Effect on high K + (80 mM)-depolarizing solution-induced contraction: As shown in the Fig.4A, sepsis (13 h post-CLP) significantly ( p <0.05) decreased the maximum aortic response to high K + -depolarizing solution (0.28 ± 0.02 g; n=10) as compared to that observed in SO mice (0.51 ± 0.01 g; n=10). However, unlike in sepsis, interestingly the maximal contractile response to high K + in the aortic rings from mice of the diabeto-septic group (0.46 ± 0.01 g, n=10) was found to be significantly ( p <0.05) higher and almost similar to that observed in mice of the SO group. Moreover, this value was significantly ( p <0.05) lower in comparison to diabetic group (0.65 ± 0.02 g; n=10). v) Effect on NA-induced contraction: Fig.4B summarizes the aortic reactivity to NA in the mice of different groups (SO, sepsis, diabetes and diabeto-sepsis). The E max and pD 2 values of NA in mice of the SO group were 0.51 ± 0.02 g and 7.45 ± 0.05 (n=8), respectively. However, in comparison to SO mice, the aortic response to NA was found to be significantly ( p <0.05) reduced (E max : 0.28 ± 0.03 g; n=6; Fig. 4B) in mice of the sepsis group (13 h post-CLP) without any change in the potency of the agonist (pD 2 : 7.32 ± 0.08 g; n=6). Interestingly, the concentration response curve of NA in diabeto-septic group was superimposed over the DRC of NA in mice of the SO group whereas the DRC of NA in diabetic group was significantly shifted towards left. The E max and pD 2 values of NA in diabeto-septic group were 0.43 ± 0.01 g and 7.41 ± 0.08 (n=8), respectively. vi) Effect on mRNA expression of α 1D -adrenoceptor in mouse aorta: To correlate the changes in aortic reactivity to NA in the mice of different groups, mRNA expression of α 1D was evaluated in the aorta of the mice of all these three groups. As illustrated in Fig.4C, sepsis significantly ( p <0.05) attenuated (0.43 ± 0.08 fold; n=4) while diabetes significantly ( p <0.05) increased (6.57 ± 0.05 fold; n=4) the aortic mRNA expression of α 1D receptors as compared to that observed in SO group (1.00 ± 0.00; n=4). However, when sepsis was induced in the mice with pre-existing diabetes, the mRNA expression of α 1D in the aorta (1.37 ± 0.21 fold; n=4) was found to be almost restored to the level as observed in SO group. In addition, the α 1D mRNA level in diabeto-septic mice was significantly ( p <0.05) higher in comparison to that observed in mice of the sepsis alone group. vii) Effect on acetylcholine-induced vasorelaxation: Compared to the maximal relaxant effect of ACh in mice of the SO group (94.58 ± 1.68 %; n=6), the maximum relaxant response to ACh was significantly ( p <0.05) reduced in aorta of the septic mouse (39.48 ± 2.20 %; n=6). However, as shown in Fig.4D, ACh-induced relaxation in aortic rings isolated from the septic mice with pre-existing diabetes was found to be significantly ( p <0.05) greater (65.78 ± 3.61 %; n=6) in comparison to that observed in the sepsis alone group. viii) Histopathological Studies Pancreas: The tissue section of pancreas from SO (Fig. 5A) groups showed normal islet of Langerhans having both beta and alpha cells. In chromium-hematoxylin-phloxine staining, beta cells take blue colour while alpha cells appear red. In diabetes groups (Fig. 5B), destruction of beta cells with vasculitis was observed, while alpha cells remain intact. In sepsis group (Fig. 5C), islet of Langerhans was found to be intact but degeneration and congestion of exocrine and acini of pancreas were observed. Further, the acini became coalesce to each other. In diabeto-sepsis group (Fig. 5D), destruction of beta cells as well as degeneration and coalesce of exocrine acini along with accumulation of edematous fluid and fibres were observed. Liver: Compared to normal histo-architecture of liver with central vein and radiating hepatic cords in SO (Fig. 6A) group, the tissue sections from diabetic group (Fig. 6B) showed congestion of central vein in hepatic parenchyma and swelling and degeneration of hepatocytes in hepatic cord with course nuclear material. In sepsis group (Fig. 6C) and diabeto-sepsis groups (Fig. 6D), liver section showed severe congestion in central vein and other parenchymal blood vessels, degeneration of hepatocytes along with infiltration of inflammatory cells in paracentral area. As summarized in Table 1, the comparative histopathological scoring based on the infiltration of inflammatory cells in liver sections from different groups was diabeto-sepsis>sepsis>diabetes= SO control. Table 1: Histopathological scoring of different organs of animals from different groups Groups Inflammatory cell infiltration in the organs Liver Lung Kidney Spleen SO 0 0 0 0 Diabetes 0 + 0 + Sepsis + ++ + ++ Diabeto-sepsis ++ +++ +++ +++ Grade 0: - negative, Grade 1 (+): 0–33% mild positive, Grade 2(++): 33–66% moderate positive, Grade 3 (+++): 66–100% severe positive. c. Lungs: Tissue section of lungs from SO group showed normal architecture of alveoli along with normal alveolar septa (Fig. 7A). In diabetes group (Fig. 7B) constriction of alveoli and thickening of alveolar walls was evident while in septic group (Fig. 7C) lung histopathology was characterized by presence of severe edema, constriction of alveoli and thickening of alveolar septa with infiltration of inflammatory cells. Lung tissue sections from diabeto-sepsis group showed constriction of alveoli and thickening of alveolar septa with congestion of alveolar capillaries and infiltration of cells (Fig. 7D). The histopathological scoring of lung tissue sections from different groups was diabeto-sepsis>sepsis>diabetes> SO control (Table 1). Kidney: In diabetes group (Fig. 8B) and sepsis group (Fig. 8C) degeneration of tubules and glomeruli, congestion in renal blood vessels with infiltration of inflammatory cells were prominent. In diabeto-sepsis group (Fig. 8D), congestion and haemorrhages with degeneration and necrosis of tubules and glomeruli, infiltration of inflammatory cells around glomeruli and tubules were comparatively higher. The order of infiltration of inflammatory cells in kidney tissue section was diabeto-sepsis>sepsis>diabetes=SO control (Table 1). Spleen: Compared to the normal orientation of red pulp and white pulp with capsule and trabeculae in SO group (Fig.9A), the tissue sections of spleen from diabetic and sepsis groups showed depletion of white pulp with necrosis of blood channels, deposition of hemosiderin pigements (Fig. 9B & C, respectively). In diabeto-sepsis group, severe congestion of sinusoids and depletion of white pulp at capsular area with excess deposition of hemosiderin pigments was observed (Fig. 9D). The histopathological scoring based on infiltration of inflammatory cells was diabeto-sepsis>sepsis>diabetes> SO control (Table 1). Discussion Major findings in the present study were i) septic mice with pre-existing diabetes died earlier in comparison to sepsis alone mice ii) vascular reactivity to NA was significantly increased in diabetic mice while decreased in septic mice as compared to control group, however, in diabeto-sepsis group NA-induced contraction was found to be similar to that observed in SO group iii) the mRNA expression of α 1D− adrenoreceptor in aorta of diabeto-septic mice was also found to be similar to that of SO mice, however, significant increase and decrease in the receptor expression were observed in diabetes and sepsis group, respectively iv) septic mice with pre-existing diabetes also exhibited marked hypoglycemia before death with reduced bacterial load and increased inflammatory cells infiltration in lungs, liver, kidney and spleen in comparison to the mice of diabetes and/or sepsis alone groups. Patho-physiology of sepsis in diabetes has been studied in number of animal models and most of these studies have primarily focused on immune system. However, impact of sepsis in pre-existing diabetes on cardio-vascular system has not attracted much attention of the scientific community. Dysregulation of cytokines functions and prolonged inflammatory response is considered to be the underlying cause of higher mortality in diabetic mice subjected to Group B Sterptococci -induced sepsis [ 13 ]. In spontaneous type-1 diabetes (T1D) as well as Goto-Kakizaki T2D model, the mortality rate in animals was enhanced following caecal ligation and puncture (CLP)-induced sepsis [ 30 , 31 ]. In addition, alloxan-induced T1D mouse was found to be highly susceptible to polymicrobial sepsis due to G-protein-coupled receptor kinase-2 (GRK2)-mediated down-regulation of chemokine receptor (CXCR-2) on blood neutrophils resulting in aberrant neutrophil migration [ 15 ]. In agreement to these observations, we also observed a significant ( p < 0.05) decrease in survival time in the mice of diabeto-sepsis group compared to the sepsis alone group in the present study. In contrast to the existing report about early mortality in complicated sepsis (sepsis in pre-existing diabetes), some research findings and epidemiological data suggest less likelihood about development of acute lung injury in septic patients with the history of diabetes as compared to the septic-patients without diabetes [ 32 , 33 ] possibly due to reduced activation of NF-κB in alveolar macrophage and decreased expression of MyD88 in lungs [ 24 ]. However, this reduced inflammatory response in diabetic rats subjected to sepsis seems to be more restricted to lungs since diabetic patients are more likely to show enhanced renal dysfunction following development of sepsis [ 33 ]. Thus, the impact of pre-existing diabetes on sepsis-induced multiple organ dysfunctions is a complex process and seems to be organ specific. In the present study, we aimed to compare the effect of pre-existing diabetes and sepsis with that of sepsis alone in respect of vascular function/reactivity. Circulatory failure in sepsis is characterized by refractory hypotension and vascular hyporeactivity to clinically used vasoconstrictors like nor-adrenaline which leads to impaired tissue perfusion and multi-organ dysfunction. Overproduction of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) along with down-regulation and desensitization of α 1D -adrenoceptor expression are considered to be the underlying mechanism of vascular hyporeactivity in sepsis [ 23 , 34 ]. Thus, we aimed to study the aortic response to NA, a commonly used vasopressor agent, in diabeto-septic mice. To our surprise, we did not observe any hyporeactivity to NA in the aortic rings from mice of the diabeto-septic group as observed in septic mice. Our observation of functional study (isometric tension measurement) is also strongly corroborated with the mRNA expression of α 1D -adrenoceptors in aortic rings where this receptor expression was found to be statistically similar in both diabeto-sepsis and SO groups. However, in consistent with the aortic reactivity to NA, mRNA expression of α 1D -adrenoceptors was down-regulated in sepsis alone group whereas up-regulated in diabetes group. Earlier report from our laboratory [ 23 ] has also demonstrated the down-regulation and desensitization of α 1D− adrenoceptors in aorta following polymicrobial sepsis in mice. But on the contrary, increase in α 1D -adrenoceptors was reported in diabetes [ 35 ]. Thus, the vascular reactivity to NA and expression of mRNA transcript of α 1D -adrenoceptor in aorta of the diabeto-septic mice in our study indicate that when diabetic mice were subjected to polymicrobial sepsis, sepsis-induced vascular hyporeactivity to NA and associated down-regulation of α 1D expression in aorta were counteracted by diabetic state of the animals. Further, similar to the aortic reactivity to NA, the maximum contractile response to high K + (indicative of voltage-gated Ca 2+ channel-dependent contraction) in diabeto-sepsis group was also found to be almost comparable to that observed in mice of SO group, while it was found to be significantly decreased in mice of the sepsis group and significantly increased in the aortic rings of diabetes group. Thus apparently, the receptor-independent and voltage-gated Ca 2+ channel-dependent arterial contractile response also remained unaltered in septic mice with pre-existing diabetes. Therefore, suggesting that diabeto-sepsis subjects are not prone to hypotensive crisis as in sepsis or hypertensive crisis as in diabetes. It has been documented that during septic shock, patients with diabetes have higher circulating levels of inflammatory biomarkers like endothelial cell adhesion (E-selectin) and vascular endothelial growth factor (sFLT-1) [ 36 ]. However, compared to sepsis alone, we did not observe any augmentation in endothelial dysfunction in the aortic rings from diabeto-septic mice as there was no significant alteration in the vasorelaxant response to ACh in the septic mice with pre-existing diabetes as compared to that in sepsis group. Thus, ruling out the possible involvement of impaired vasorelaxant response in sepsis-induced reduction in survival time in pre-existing diabetic mice. Hypoglycemia results in poor outcome in diabetic patients [ 37 , 38 ]. On the other hand, septic patients are often encountered with stress-induced hyperglycemia [ 39 ], however, the severity of hyperglycemia and nature of critical illness determine the prognosis in septic patients [ 40 ]. Hyperglycemia in sepsis is not only a marker of severity but also governs the adverse outcome in different vital organs during sepsis [ 41 , 42 ]. It disrupts the functions of innate immunity by reducing neutrophil chemotaxis, formation of reactive oxygen species and phagocytosis of bacteria despite accelerated infiltration of leukocytes into the peripheral tissues [ 43 , 44 ]. But numerous studies have reported an association between hypoglycemia and high mortality in severely ill septic patients [ 45 – 47 ]. The hypoglycemic state in sepsis is thought to be due to cytokine-mediated reduction in glucose production [ 48 , 49 ] and down-regulation of GLUT2 [ 50 ]. Hypoglycemia in sepsis may develop despite increased glucose production and is mediated by non-insulin dependent increased uptake and utilization of glucose in tissues rich in macrophages like liver, lung, spleen and ileum [ 51 , 52 ]. Consistent with these reports, we also observed an early hyperglycemic stage followed by euglycemia and hypoglycemia in septic mice whereas early hyperglycaemia followed by hypoglycaemic phase in diabeto-septic mice. On histopathological examination, marked infiltration of inflammatory cells was observed in macrophage-rich tissues like liver, lung, kidney and spleen of the mice of diabeto-sepsis group. Apart from this, significant decrease in total bacterial counts was also observed in the peritoneal lavage (source of infection) and blood (systemic infection) of the mice of diabeto-sepsis group. Thus, suggesting that heavy infiltration of inflammatory cells in different tissues reduces the bacterial load in this group of mice and is responsible, at least in part, for reduction in blood glucose level leading to hypoglycemic shock in diabeto-septic subjects. Conclusion Taken together, based on the findings of present study, it may be inferred that the early mortality in septic mice with pre-existing diabetes (in comparison to sepsis alone) possibly seems to be due to increased infiltration of inflammatory cells and associated hypoglycemia (non-insulin dependent) rather than vascular dysfunction as observed in sepsis. Declarations Funding: The research work is not supported by any grants from specific Funding Agency. However, financial assistance under the “Strengthening and Development Grant to the University” by the Indian Council of Agricultural Research, New Delhi, for support of routine research work of postgraduate students is thankfully acknowledged. The laboratory facility established by the Department of Veterinary Pharmacology and Toxicology, DUVASU, Mathura, India, under ICAR-sponsored Niche Area of Excellence Programme (Grant No. 10 (10)/2012-EPD dated 23rd march 2012) is also thankful acknowledged. Conflict of interest: None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper. Author contribution: MG conducted the functional study, MG, TJ and SC conducted the mRNA expression study, MG, AS, NG performed the histopathological experiments, AS, SC, MG analyzed the data, SC and SKG conceptualized the experiments and wrote the MS. All the authors have read and approved the manuscript. References Bosmann, M., & Ward, P. A. (2013). The inflammatory response in sepsis. Trends in immunology , 34 (3), 129–136. https://doi.org/10.1016/j.it.2012.09.004 Angus, D. C., Linde-Zwirble, W. T., Lidicker, J., Clermont, G., Carcillo, J., & Pinsky, M. R. (2001). 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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-3857212","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266818641,"identity":"de7360fd-fb33-4761-a199-a92411bf2409","order_by":0,"name":"Manju Gari","email":"","orcid":"","institution":"Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU)","correspondingAuthor":false,"prefix":"","firstName":"Manju","middleName":"","lastName":"Gari","suffix":""},{"id":266818642,"identity":"f5c2ed7c-4cc8-4e94-85fd-b8d9ab1125be","order_by":1,"name":"T Jagadeesh","email":"","orcid":"","institution":"Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU)","correspondingAuthor":false,"prefix":"","firstName":"T","middleName":"","lastName":"Jagadeesh","suffix":""},{"id":266818643,"identity":"b4054100-171f-412b-b7f4-4cf5bee5963b","order_by":2,"name":"Soumen Choudhury","email":"","orcid":"","institution":"Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU)","correspondingAuthor":false,"prefix":"","firstName":"Soumen","middleName":"","lastName":"Choudhury","suffix":""},{"id":266818644,"identity":"553f8515-3ff6-4ae4-8fd5-aafd3ef7a5d8","order_by":3,"name":"Amit Shukla","email":"","orcid":"","institution":"Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU)","correspondingAuthor":false,"prefix":"","firstName":"Amit","middleName":"","lastName":"Shukla","suffix":""},{"id":266818645,"identity":"8b4fc4c9-1ccd-464a-bd97-4aaa3e625882","order_by":4,"name":"Neeraj K Gangwar","email":"","orcid":"","institution":"Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU)","correspondingAuthor":false,"prefix":"","firstName":"Neeraj","middleName":"K","lastName":"Gangwar","suffix":""},{"id":266818646,"identity":"4f01f4d7-8722-458a-af6f-3168b986aa89","order_by":5,"name":"Satish K Garg","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYJCCD0DMwyDBfABIS8gQo4NxBgODAVALWwJICw/RWkCKDRjA1hEC5tKHDzb+qPkjIz+75/OrGzUWPAzsh49uwKfFsi8tsZnnmAGPwZ2z26xzjgEdxpOWdgOfFoMzPOaPGdiAWiRytxnnsAG1SPCYEdJi2PjjnwGP/IycZ8Y5/4jU0sDbBgyxGznMj3PbiNBi2cOW2MzbZ8xjcCPNjDm3T4KHjZBfzHmYgSH2Tc5efkby48853+rk+NkPH8PvMCQ2mwSYxKccXQvzB0KqR8EoGAWjYGQCAPkAQtmKNCCuAAAAAElFTkSuQmCC","orcid":"","institution":"Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU)","correspondingAuthor":true,"prefix":"","firstName":"Satish","middleName":"K","lastName":"Garg","suffix":""}],"badges":[],"createdAt":"2024-01-12 14:14:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3857212/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3857212/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49714880,"identity":"fa7a69b5-3b41-4c1d-b8c6-72e622d1c255","added_by":"auto","created_at":"2024-01-16 21:01:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":504842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of diabetes on vascular reactivity in mice. \u003c/strong\u003e\u0026nbsp;Induction of diabetes significantly increased high K\u003csup\u003e+\u003c/sup\u003e (80 mM)-depolarizing solution induced contraction in mouse aorta after 8\u003csup\u003eth\u003c/sup\u003e weeks of STZ administration (A). Line diagrams (B) showing concentration-dependent contractile response of mouse aorta to nor-adrenaline (NA). Significant increase in the maximum contraction of NA was achieved following 8\u003csup\u003eth\u003c/sup\u003e week of STZ administration in diabetic rats in comparison to healthy control. Data are presented as mean ± SEM; n=6-8. Vertical bars represent SEM. Mean values of KDS-induced contraction between two groups were analyzed by unpaired student’s ‘t’ test. Concentration-dependent agonist response data were analyzed by two-way ANOVA followed by Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 \u003cem\u003evs.\u003c/em\u003e healthy control;\u0026nbsp; #\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 \u003cem\u003evs.\u003c/em\u003e diabetes (5\u003csup\u003eth\u003c/sup\u003e week).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/29c404abb7388f2ecfa59d3e.png"},{"id":49713088,"identity":"2cf9b305-d9b7-4b3f-867e-18f6826492e9","added_by":"auto","created_at":"2024-01-16 20:37:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":394951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of sepsis on vascular reactivity in mice. \u003c/strong\u003ePolymicrobial sepsis induced by caecal ligation and puncture (CLP) significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) reduced the survival time (18.15 ± 0.18 h; n=11) in septic mice (A). All the animals from sham-operated (SO) group survived up to 72 h of observation period. Sepsis significantly reduced aortic contraction to high K\u003csup\u003e+\u003c/sup\u003e (80 mM) depolarizing solution at 18h post CLP as compared to SO mice (B). Cumulative concentration-dependent contractile response curves to nor-adrenaline (NA) showing significant reduction in the NA-induced maximum contraction in septic mice (18h post CLP) in comparison to sham-operated mice (C).\u0026nbsp; Data are presented as mean ± SEM; n=8-11. Vertical bars represent SEM. The overall difference in survival rate was determined by the Kaplan-Meier test followed by the log-rank test. Mean values of KDS-induced contraction between two groups was analyzed by unpaired student’s t test. Concentration-dependent agonist response data were analyzed by two-way ANOVA followed by Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test. Statistical significance was considered as *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/73217f8d26953c4d4cf3dacb.png"},{"id":49713087,"identity":"a00db03c-6808-4413-92e5-cb33787dc8aa","added_by":"auto","created_at":"2024-01-16 20:37:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":674655,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curves (A) showing the comparative survival time in different groups of animals. Induction of sepsis\u003cstrong\u003e \u003c/strong\u003ein mice with pre-existing diabetes reduced the survival time (13.23 ± 0.36 h; n=9) as compared to the septic mice (18.15 ± 0.18 h; n=11). Line diagrams (B) showing the different phases of glucose level in blood following induction of sepsis in different groups of animals. Early hyperglycaemic phase (2-10 h post CLP), followed by euglycaemic phase (12-16 h post CLP) and late hypoglycaemic phase (18-20 h post CLP) was observed in septic mice whereas in diabeto-sepsis mice only early hyperglycaemia (2-10 h post CLP) followed by sharp hypoglycaemic phase (12-14 h post CLP) was seen. Diabetic mice showed consistently higher blood glucose level. The Scattered plots showing the effect of sepsis on bacterial load in blood (C) and peritoneal lavage (PL; D) of the mice with pre-existing diabetes. Note that significant reduction in the bacterial load in both blood and peritoneal lavage were observed in diabeto-sepsis group in comparison to sepsis alone group. Data are presented as mean ± SEM. Vertical bars represent SEM. The overall difference in survival rate was determined by the Kaplan-Meier test followed by the log-rank test. The total bacterial loads, expressed as bacterial colony-forming units (CFU), between different groups were compared by using the nonparametric Kruskal–Wallis test followed by a post-hoc Dunn's test. Statistical significance was considered as *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/d8986496945e15169b4471ab.png"},{"id":49713090,"identity":"5deecf1f-914c-4e73-ba7e-4556655f1455","added_by":"auto","created_at":"2024-01-16 20:37:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":768515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of sepsis on vascular reactivity in the mice with pre-existing diabetes. \u003c/strong\u003e(A) Bar diagram showing the effect on high K\u003csup\u003e+ \u003c/sup\u003e(80 mM) depolarizing solution (KDS)-induced contraction. Note that, sepsis (13h post CLP) significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) reduced KDS-induced maximum contraction while diabetes enhanced this aortic response as compared to sham-operated group. (B) Comparative line diagrams showing concentration-dependent contractile responses to NA in mouse aorta from different groups. Though, sepsis (13 h post CLP) significantly reduced the aortic response to NA as compared to that observed in sham-operated mice, aortic reactivity to NA in the septic mice with pre-existing diabetes were comparable to sham-operated mice. Significant increase in NA-induced contraction was observed in the aorta of diabetic mice. (C) Bar diagrams showing the comparative mRNA expression of α1D-adrenoceptors in mouse aorta from different groups. Unlike sepsis alone group, induction of sepsis in pre-existing diabetic mice did not produce any significant change in the mRNA expression of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptors in mouse aorta in comparison to sham-operated group. (D) Comparative line diagrams showing concentration-dependent relaxant response to ACh in mouse aorta from different groups. Data are presented as mean ± SEM; n=6-11. Vertical bars represent SEM. Mean values of KDS-induced contraction or mRNA expression of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptors between groups were analyzed by one-way ANOVA followed by Tukey’s \u003cem\u003epost-hoc\u003c/em\u003e test. Concentration-dependent agonist response data were analyzed by two-way ANOVA followed by Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test. Statistical significance was considered at *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/377dd4683a5e762a16abea74.png"},{"id":49713089,"identity":"d41232ce-4293-47bd-bcac-9d4a96b01fa8","added_by":"auto","created_at":"2024-01-16 20:37:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":982135,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative photomicrographs of Chromium-hematoxylin-phloxine stained pancreatic tissue sections from different groups.\u003c/strong\u003e Tissue section of pancreas from SO mice (A) shows normal islet of Langerhans having both \u003cem\u003ebeta \u003c/em\u003e(stained blue) and \u003cem\u003ealpha\u003c/em\u003e (stained red) cells. In diabetes groups (B), destruction of \u003cem\u003ebeta\u003c/em\u003ecells with vasculitis was observed, while \u003cem\u003ealpha\u003c/em\u003ecells remain intact. In sepsis group (C), islet of Langerhans was found to be intact but degeneration and congestion of exocrine and acini of pancreas were observed. In diabeto-sepsis group (D), destruction of \u003cem\u003ebeta\u003c/em\u003ecells as well as degeneration and coalesce of exocrine acini along with accumulation of edematous fluid and fibres were observed.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/4f5e760b1b350644cabd1917.png"},{"id":49713094,"identity":"5ddd4caa-620c-42f1-a2ba-312e97b7f9e8","added_by":"auto","created_at":"2024-01-16 20:37:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":999895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative photomicrographs of H\u0026amp;E stained liver tissue sections from different groups.\u003c/strong\u003e Liver tissue section from SO mice (A) shows normal histo-architecture of hepatocytes and central vein. Diabetes group (B) shows swelling and vacuolation of hepatocytes with coarse nuclear material and congestion of central vein. Tissue sections from sepsis group (C) showing severe congestion of central veins, nuclear karyomegaly, fragmentation and infiltration of inflammatory cells. The diabeto-sepsis (D) group shows severe congestion of central veins, nuclear karyomegaly, fragmentation and infiltration of inflammatory cells. \u0026nbsp;Bars represent\u003c/p\u003e\n\u003cp\u003e50 μm length. 200x H\u0026amp;E.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/54656ba293d203c19a11ce88.png"},{"id":49714612,"identity":"1a313419-abb2-4466-a2a2-c313eb6193b7","added_by":"auto","created_at":"2024-01-16 20:53:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1024698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative photomicrographs of H\u0026amp;E stained lung tissue sections from different groups. \u003c/strong\u003eLung tissue section from SO mice (A) shows normal architecture of alveoli along with normal alveolar septa. Diabetes group (B) shows constriction of alveoli and thickening of alveolar walls. Tissue sections from sepsis group (C) showing severe edema, constriction of alveoli and thickening of alveolar septa with infiltration of cells. The diabeto-sepsis group (D) showing constriction of alveoli and thickening of alveolar septa with congestion of alveolar capillaries and infiltration of cells. Bars represent 50 μm length. 400x H\u0026amp;E.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/f55b7310bc05ee3e5fa7885b.png"},{"id":49713989,"identity":"9f17fc4c-7301-4983-9800-092e2f88949c","added_by":"auto","created_at":"2024-01-16 20:45:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1133641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative photomicrographs of H\u0026amp;E stained kidney tissue sections from different groups. \u003c/strong\u003eKidney tissue section from SO mice (A) shows normal histoarchitecture of tubules and glomerulous while diabetes group (B) shows degeneration and swelling of tubular epithelium. Sespis group (C) shows congestion with mild degeneration of tubules and glomeruli and infiltration of inflammatory cells and diabeto-sepsis group (D) show severe infiltration of polymorphonuclear cells with congestion of blood vessels and degeneration of glomeruli, renal tubules. Bars represent 50 μm length. 400x H\u0026amp;E.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/d1f904957f86d7f741791956.png"},{"id":49713093,"identity":"66367c87-bb55-4775-ab55-3280d41ec7aa","added_by":"auto","created_at":"2024-01-16 20:37:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1375656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative photomicrographs of H\u0026amp;E stained spleen tissue sections from different groups. \u003c/strong\u003eSpleen tissue section from SO mice (A) shows normal histoarchitecture of red and white pulp with trabeculae. Diabetes group (B) shows depletion of white pulp with necrosis of sinusoids and deposition of hemosiderin pigment. Tissue sections from sepsis group (C) shows congestion of sinusoids and mild depletion of white pulp at periphery and diabeto-sepsis (D) group shows severe congestion of sinusoids, depletion of white pulp at periphery with deposition of yellowih golden colour hemosiderin pigment. Bars represent 50 μm length. 400x H\u0026amp;E\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/779aa48ab7687f79f6287dac.png"},{"id":54599523,"identity":"652a3fb9-6df6-4ddc-996d-b2bf0e4d7081","added_by":"auto","created_at":"2024-04-12 20:39:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7874137,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/c7b16960-661f-4649-81a6-7bc76c215c28.pdf"},{"id":49713086,"identity":"07b9be1b-39fa-4049-b47f-6697c5995bcc","added_by":"auto","created_at":"2024-01-16 20:37:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18180,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3857212/v1/b4d5befb8970329b263985f1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hypoglycemia rather than vascular dysfunction causes early mortality in diabeto-septic mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is a multifactorial disease with uncontrolled systemic production of inflammatory mediators (\u0026lsquo;cytokine storm\u0026rsquo;) that leads to systemic inflammatory response syndrome (SIRS) following systemic microbial infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Sepsis ranks tenth leading cause of deaths in the United States with mortality rates varying between 30 and 70% among ICU patients [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Sepsis is often complicated by other co-morbidity conditions especially cardiovascular disorders and diabetes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Local and systemic (sepsis) infections are frequently encountered in patients with diabetes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and regardless of the type, diabetic patients have higher risks of infection [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As per the available information, approximately 22% mortalities in diabetic human patients are associated with infections [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In experimental animals too, diabetic mice have been reported to be highly susceptible to polymicrobial sepsis [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, most of the preclinical sepsis studies in laboratory animals have been typically conducted in healthy animals without considering the clinical relevance or impact of co-morbidities due to diabetes and sepsis.\u003c/p\u003e \u003cp\u003eHyperglycemia, abnormalities in renin-angiotensin axis, enhanced vascular smooth muscle contraction, endothelial dysfunctions, and nephropathy are some of the reasons for the development of hypertension in diabetes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. On the other hand, circulatory failure in septic patients is mainly due to hypotension leading to hypo-perfusion of majority of the body organs. Therefore, in a pathological situation, where pre-existing hypertension (like in diabetes) precedes the hypotensive state (like in sepsis), a more realistic and complex clinical condition is likely when diabetes and sepsis co-exist, and thus, vascular reactivity is likely to be affected in a different way. Numerous studies have documented an increase in the sensitivity of vascular smooth muscles to noradrenaline (NA) in arteries from diabetic animals [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] while vascular hyporeactivity to vasoconstrictors including NA is considered to be the underlying cause of death in sepsis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thus, we aimed to study the effect of sepsis on vascular dysfunction in pre-existing diabetes mice model in an endeavour to see whether the death of diabeto-sepsis animals occurs because of vasoplegia or some other reason.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003eAdult male Swiss albino mice (26\u0026ndash;28 g) were procured from Disease Free Small Animal House, College of Veterinary and Animal Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, and kept in polypropylene cages in the Departmental Laboratory Animal House under 12\u0026ndash;12 h dark-light cycle. Experimental animals had free access to pellet feed (Ashirwad Industries, Mohali, Punjab) and drinking water. An acclimatization period of 10 days was allowed before starting the actual study. The study was undertaken after obtaining approval from the Institutional Animal Ethics Committee (IAEC) of the University (Approval No.: IAEC/17/12, Dated 26-07-2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDrugs and chemicals\u003c/h2\u003e \u003cp\u003eStreptozotocin (STZ), acetylcholine (ACh), nor-adrenaline (NA), and phenylephrine (PE) were procured from Sigma-Aldrich (St. Louis, Mo, USA). All other used chemicals/reagents were of analytical grade. Noradrenaline (NA) was dissolved in 0.1 N hydrochloric acid plus 0.01% ascorbic acid. Streptozotocin was dissolved in 0.1 M citrate buffer solution. All other chemicals were dissolved in distilled water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eInduction of diabetes\u003c/h2\u003e \u003cp\u003eType-1 diabetes in mice was induced by administering five sequential daily intra-peritoneal injections of freshly prepared streptozotocin solution (STZ @ 65 mg/kg b.w.) as described earlier [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Streptozotocin solution was prepared immediately before use and injected within 5 min of dissolution as STZ degrades within 15\u0026ndash;20 min after dissolving in citrate buffer. Body weights of animals and blood glucose levels were monitored at the weekly interval to assess the progression of diabetes. Animals showing\u0026thinsp;\u0026gt;\u0026thinsp;300 mg/dl blood glucose levels were considered to be diabetic. 10% sucrose solution in water was provided to mice by oral route to avoid hypoglycemia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInduction of sepsis\u003c/h2\u003e \u003cp\u003eSepsis was induced by the caecal ligation and puncture method (CLP) in mice as described earlier [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, following overnight fasting, mice were anesthetized with xylazine (10 \u0026micro;g/g b.wt, \u003cem\u003ei.p\u003c/em\u003e) and ketamine (80 \u0026micro;g/g b.wt, \u003cem\u003ei.p.\u003c/em\u003e). A midline incision (2cm long) was made in the abdominal region to expose the cecum and thereafter the cecum was ligated with 2\u0026thinsp;\u0026minus;\u0026thinsp;0 silk distal to the ileocecal valve to avoid intestinal obstruction. The cecum was then punctured twice with a 21G needle and placed back into the abdominal cavity. Then the abdominal incision was closed in layers. To prevent dehydration, isotonic sodium chloride solution (1 mL/mouse) was subcutaneously injected to all the mice. Sham-operated (SO) mice were subjected to the same surgical procedure except CLP, and served as control. After induction of sepsis or only surgical procedure in mice of SO group, animals were closely observed for up to 72 h for development of sepsis based on lethargy, induction of conjunctivitis, absence of grooming behavior, ruffled fur, and reduced feed and water intake or any other apparent change in behavior of animals including mortality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInduction of diabeto-sepsis\u003c/h2\u003e \u003cp\u003eIn a separate group of mice, first diabetes was induced by administering STZ as mentioned above and after ensuring the development of diabetes (eight weeks after administration of the first dose of STZ), the animals were subjected to caecal ligation and puncture to induce sepsis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSurvival times:\u003c/h2\u003e \u003cp\u003eAnimals of all the four groups were closely observed for any signs of discomfort or survival for up to 72 hours. The survival curves and mean survival times for mice of all the groups were plotted by Kaplan-Meier survival curve method and analyzed using log-rank test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of total bacterial load\u003c/h2\u003e \u003cp\u003eTotal bacterial load in blood (systemic infection) and peritoneal fluid (source of infection) were determined as mentioned earlier [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] for assessment of sepsis. Briefly, blood samples were collected by cardiac puncture under xylazine-ketamine anesthesia. For the collection of the peritoneal lavage (PL), 2 ml sterile phosphate buffer saline (PBS) was injected into the peritoneal cavity at the time of sacrifice of animal and PL was aseptically collected after giving an incision in the abdominal region and before collecting the organs of interest for other studies. The samples (blood and PL) were then serially diluted (1:10) in sterile phosphate buffer saline (PBS) and 100\u0026micro;l of each dilution was plated on Luria-Bertani agar plates and incubated at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 18 h. Each dilution was plated in duplicate and bacterial loads were expressed as log CFU/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCollection of tissues for functional and molecular studies:\u003c/h2\u003e \u003cp\u003eMice of the diabetes, sepsis, diabeto-sepsis and SO groups were sacrificed by bleeding from vena cava under xylazine-ketamine anaesthesia after 18 h of the CLP or SO procedure. Thorax and abdomen were cut open and lungs and heart were taken out en-bloc along with the thoracic aorta and immediately placed in ice-cold (4\u003csup\u003e\u0026deg;\u003c/sup\u003eC) Modified Krebs-Henseleit solution (MKHS- 118.0 NaCl, 4.7 KCl, 2.5 CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 1.2 MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 1.2 KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 11.9 NaHCO\u003csub\u003e3,\u003c/sub\u003e and 11.1 glucose mmol/L). Thoracic aorta was cleaned off the adhering connective tissues under a dissection stereo- microscope (Motic, China) and aortic rings of 3\u0026ndash;4 mm length were prepared without damaging the endothelium. To study the endothelium-independent response of aortic rings, endothelial denudation was undertaken by passing horse tail hair through the arterial rings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of isometric tension\u003c/h2\u003e \u003cp\u003eAortic rings from the mice of different treatment groups were mounted between two \u0026ldquo;L\u0026rdquo; shaped hooks made from 37 G stainless steel wire and mounted under a resting tension of 1.0 g in thermostatically controlled (37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003e\u0026deg;\u003c/sup\u003eC) organ bath (Radnoti, USA) of 10 ml capacity containing modified Krebs\u0026ndash;Henseleit solution (MKHS) continuously bubbled with medical gas (74% N\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;21% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;5% CO\u003csub\u003e2\u003c/sub\u003e). Isometric tension was measured using the high-sensitivity isometric force transducer and recorded in a PC using LabChart V6.1.3 Pro software programme (Powerlab, AD Instruments, Australia). Before starting any experimental protocol, tissue viability was assessed by recording the aortic contraction to a high K\u003csup\u003e+\u003c/sup\u003e (80 mM) depolarizing solution.\u003c/p\u003e \u003cp\u003eNor-adrenaline is commonly used as a vasopressor agent in septic patients and it also has a role in hypertension in diabetic patients. Therefore, cumulative concentrations response to NA (0.1 nM \u0026ndash; 10 \u0026micro;M) at an increment of 0.5 log concentration unit was studied in the arterial rings of the mice from different groups. In a separate set of experiment, to assess the alterations in endothelium-dependent relaxant responses of the aortic rings, concentration-dependent responses to acetylcholine (ACh; 1 nM\u0026ndash;10 \u0026micro;M) was also recorded in phenylephrine (PE)-pre-contracted aortic rings from the mice from different groups. Before eliciting the relaxant response to ACh in the aortic rings from mice of different groups, the matching mean pre-contractile tensions (0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g) were generated in all the tissues using phenylephrine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcriptase polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThoracic aorta was collected in 0.1% diethyl pyrocarbonate (DEPC) treated autoclaved phosphate buffer saline (PBS), and after removing the adjacent fat and connective tissues, samples were quickly snap-frozen in liquid nitrogen and stored in RNA later at \u0026minus;\u0026thinsp;80\u0026deg;C until further use. Total RNA was isolated using commercially available kit (Ambion, Thermo scientific) by following the manufacturer\u0026rsquo;s protocol. Samples were then treated with RNase-free DNase and DNase was subsequently inactivated by heating at 56\u0026deg;C for 10 min and immediately chilled at 4\u0026deg;C. The purity of RNA was checked by biophotometer (Eppendorf, USA). cDNA synthesis (from 150 ng total RNA) was carried out using Revertaid\u0026reg; First strand cDNA synthesis kit (Thermo Scientific, USA) using Moloney murine leukemia viral reverse transcriptase enzyme by following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eqRT-PCR reactions were performed in duplicate using SYBR Green chemistry (PowerUp\u003csup\u003eTM\u003c/sup\u003eSYBR\u0026trade; Green master mix [2X]; ThermoFischer Scientific, USA) in QuantStudio 3, Applied Biosystems). Each reaction was consisted of SYBR Green master mix (5 \u0026micro;l), gene-specific forward and reverse primers (0.5 \u0026micro;L each of 10 pmol/\u0026micro;L stock) and cDNA (1 \u0026micro;l) in a total volume of 10 \u0026micro;L. The real-time PCR reaction was started with initial incubation at 95\u0026deg;C for 10 min followed by 42 cycles of amplification with denaturation at 95\u0026deg;C for 1 min, annealing at (temperature as mentioned below) for 1 min and extension at 72\u0026deg;C for 1 min each. The optimum annealing temperature determined by PCR for α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptor specific primer sets (F5\u0026prime;-GCCTCTGAGGTGGTTCTGAG-3\u0026prime;, R 5\u0026prime;-GGACGAAGAAAAAGGGGAAC-3\u0026prime;; 208 bp) was 57\u0026deg;C and for GAPDH (the reference gene- F 5\u0026prime;-AACTTTGGCATTGTGGAAGG-3\u0026prime;, R 5\u0026prime; ACACATTGGGGGTAGGAACA-3\u0026prime;; 223 base pairs) was 58\u0026deg;C. To assess the specificity of the amplified product, dissociation curve was generated at temperature of 60\u0026deg;C through 95\u0026deg;C. The results were expressed as threshold cycle values (C\u003csub\u003eT\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological studies:\u003c/h2\u003e \u003cp\u003ePancreas, liver, lungs, kidney, and spleen were collected from animals of all the four groups (SO, diabetes, sepsis and diabeto-sepsis) in 10% buffered formal saline and kept for 72 h for fixation. Tissue sections of approximately 4\u0026ndash;5 \u0026micro;m thickness were prepared and stained using Hematoxylin and Eosin (H \u0026amp; E) stain and examined under light microscope (Nikon, Japan) to determine the pathological lesions in different organs of the mice of different groups. Special staining of the pancreatic sections was undertaken using Gomori\u0026rsquo;s Chromium-hematoxylin-phloxine stain for determining the extent of destruction of \u003cem\u003ebeta\u003c/em\u003e cells of the pancreas [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The scoring of histopathological lesions was done as described earlier [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and \u0026lsquo;n\u0026rsquo; refers to the number of animals used in different experimental protocols. The overall difference in survival rate was determined by the Kaplan\u0026ndash;Meier test followed by the log-rank test. For measurement of the bacterial colony-forming units (CFU), all data points are presented. Groups were compared by using the nonparametric Kruskal\u0026ndash;Wallis test followed by a post-hoc Dunn's test.\u003c/p\u003e \u003cp\u003eThe E\u003csub\u003emax\u003c/sub\u003e (the maximal response) and the EC\u003csub\u003e50\u003c/sub\u003e (the concentration producing 50% of the maximal response) values were determined by non-linear regression analysis using GraphPad Prism V.4.00 (San Diego, California). Potency is defined as the pD\u003csub\u003e2\u003c/sub\u003e value which is the -log of EC\u003csub\u003e50\u003c/sub\u003e value. Concentration-dependent agonist response data were analyzed by two-way ANOVA followed by Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test by using GraphPad Prism.\u003c/p\u003e \u003cp\u003eTo study the relative change in gene expression, the 2\u003csup\u003e\u0026minus;∆∆C\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e method was used as described earlier [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The formula used to calculate the \u0026ldquo;fold change in gene expression\u0026rdquo; was =\u0026thinsp;2\u003csup\u003e\u0026minus;∆∆C\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e,\u0026rdquo; [where ∆∆C\u003csub\u003eT\u003c/sub\u003e = (C\u003csub\u003eT,target gene\u003c/sub\u003e - C\u003csub\u003eT,GAPDH\u003c/sub\u003e) treatment - (C\u003csub\u003eT,target gene\u003c/sub\u003e -C\u003csub\u003eT,GAPDH\u003c/sub\u003e) control]. The gene-specific amplification was corrected for the difference in input of RNA by taking house-keeping gene GAPDH to account. For diabetes, sepsis and diabeto-sepsis groups, evaluation of 2\u003csup\u003e\u0026minus;∆∆C\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e indicates the fold change in gene expressions relative to SO control (SO control\u0026thinsp;=\u0026thinsp;1). The results were analyzed in comparison with the C\u003csub\u003eT\u003c/sub\u003e (minimum threshold of amplification) value of the target gene and the reference gene (GAPDH). Difference in values was considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eA) Assessment of hyperglycemia and associated vascular dysfunctions in diabetic mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei) Effect on blood glucose levels and body weight gain:\u0026nbsp;\u003c/strong\u003eBlood glucose levels were measured at weekly interval and the data are summarized in Table S-1 (Supplementary file). Mean blood glucose level of \u0026gt; 300 mg/dl (hyperglycemia) was observed in animals of the diabetes group from 3\u003csup\u003erd\u003c/sup\u003e week onwards following STZ administration and this hyperglycemic level was maintained up to 8\u003csup\u003eth\u003c/sup\u003e week of observation period. Compared to the body weights of animals of both the groups (healthy control and diabetes) on day 0, body weight gain after eight weeks in mice of the diabetes group was significantly lower than in the animals of healthy control group (Supplementary Table S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eii)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEffect on high K\u003csup\u003e+\u003c/sup\u003e (80 mM)-depolarizing solution-induced contraction:\u0026nbsp;\u003c/strong\u003eGiven that\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003evascular dysfunction(s) are late manifestation of diabetes, we compared the vascular reactivity to different spasmogens and relaxant agents in aortic rings of the diabetic mice sacrificed after 5\u003csup\u003eth\u003c/sup\u003e and 8\u003csup\u003eth\u003c/sup\u003e weeks of STZ administration. As shown in Fig. 1A the maximal contraction induced by high K\u003csup\u003e+\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(80 mM)-depolarizing solution (KDS) was found to be significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) higher (0.65 \u0026plusmn; 0.02 g, n=10)\u0026nbsp;after 8\u003csup\u003eth\u003c/sup\u003e week of STZ administration as compared to that observed in healthy control group (0.50 \u0026plusmn; 0.02 g; n=10). However, High K\u003csup\u003e+\u003c/sup\u003e-induced contraction was found to be statistically similar in the diabetic mice after 5\u003csup\u003eth\u003c/sup\u003e week of STZ administration (0.56 \u0026plusmn; 0.03 g, n=10) as compared to that observed in healthy control group (0.50 \u0026plusmn; 0.02 g; n=10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiii) Effect on noradrenaline (NA)-induced contraction:\u0026nbsp;\u003c/strong\u003eNoradrenaline (NA) produced concentration-dependent (0.1 nM \u0026ndash; 10 \u0026micro;M) contraction in aortic rings of the mice of healthy control group. As shown in Fig.1B,\u0026nbsp;the cumulative concentration-response curve of NA in diabetes group after 8\u003csup\u003eth\u003c/sup\u003e week of STZ administration was significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) shifted towards left with increase in the maximal contraction as compared to healthy control (E\u003csub\u003emax\u003c/sub\u003e:\u0026nbsp;0.75 \u0026plusmn; 0.04 g \u003cem\u003evs.\u003c/em\u003e0.51 \u0026plusmn; 0.04 g, n=8). However, no significant change was observed in the maximal contraction of NA in 5\u003csup\u003eth\u003c/sup\u003e week diabetic group (0.53 \u0026plusmn; 0.03 g; n=8) in comparison to healthy control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB) Assessment of vascular dysfunctions in septic (CLP) mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei) Effect on survival time:\u0026nbsp;\u003c/strong\u003eFig.2A shows the survival curves and mean survival times of the mice of sepsis (CLP) and sham-operated (SO) groups. All the mice of SO group (n=15) survived during the observation period of 72 h while the mean survival time in mice of the sepsis group (18.15 \u0026plusmn; 0.18 h; n=11) was found to be significantly (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt;0.001) lower.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eii) Effect on bacterial load:\u0026nbsp;\u003c/strong\u003eNo bacterial colonies were observed either in blood or in the peritoneal lavage of the mice from SO group. However, comparatively higher total bacterial load was observed in the blood (6.58 \u0026plusmn; 0.26 Log CFU/ml, n=6) and peritoneal lavage (10.44 \u0026plusmn; 0.13 Log CFU/ml, n=6) of the mice from septic group in comparison to SO group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eiii)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEffect on high K\u003csup\u003e+\u003c/sup\u003e (80 mM)-depolarizing solution-induced contraction:\u0026nbsp;\u003c/strong\u003eAs illustrated in Fig. 2B, sepsis significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) attenuated the maximum contraction elicited by 80 mM KDS (0.27\u0026nbsp;\u0026plusmn; 0.01 g; n=10)\u0026nbsp;in the aortic rings after 18h of CLP as compared to that observed in SO group (0.51 \u0026plusmn; 0.01 g, n=10). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiv) Effect on NA-induced contraction:\u003c/strong\u003e As shown in Fig. 2C, in comparison to the aortic response in mice of SO group,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esepsis significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) reduced the maximal contractile response to NA in the aortic rings isolated 18h post-CLP (E\u003csub\u003emax\u003c/sub\u003e: 0.51\u0026nbsp;\u0026plusmn; 0.02 g\u0026nbsp;\u003cem\u003evs.\u003c/em\u003e 0.24 \u0026plusmn; 0.02 g; n=8) but without any change in the potency (pD\u003csub\u003e2\u003c/sub\u003e: 7.45\u0026nbsp;\u0026plusmn; 0.05\u0026nbsp;\u003cem\u003evs.\u0026nbsp;\u003c/em\u003e7.43 \u0026plusmn; 0.07, n=8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC) Effect of sepsis on vascular function in pre-existing diabetic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei) Effect on survival time:\u003c/strong\u003e As shown in the Fig.3A, septic mice with pre-existing diabetes (diabeto-septic group) showed early mortality (mean survival time: 13.23 \u0026plusmn; 0.36 h; n=9) as compared to the septic mice (18.15 \u0026plusmn; 0.18 h; n=11). Thus, in further study to compare the vascular reactivity of the mice between sepsis and diabeto-sepsis groups, mice were sacrificed 13 h post-CLP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eii) Effect on blood glucose level:\u0026nbsp;\u003c/strong\u003eTime-dependent progression of blood glucose level after induction of sepsis was measured and illustrated in Fig.3B. In septic mice, blood glucose level exhibited three different phases, namely- early hyperglycaemic phase (2-10 h post CLP), followed by euglycaemic phase (12-16 h post CLP) and late hypoglycaemic phase (18-20 h post CLP) whereas the septic mice with pre-existing diabetes showed only early hyperglycaemia (2-10 h post CLP) followed by sharp hypoglycaemic phase (12-14 h post CLP). Diabetic mice showed consistently higher blood glucose level.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiii) Effect on bacterial load:\u0026nbsp;\u003c/strong\u003eAs shown in Fig. 3C\u0026amp;D, in the septic mice with pre-existing diabetes, total bacterial count was comparatively lower in blood (5.26 \u0026plusmn; 0.19 Log CFU/ml, n=6) and peritoneal lavage (9.15 \u0026plusmn; 0.27 Log CFU/ml, n=6) as compared to the respective counts in the septic mice (6.58 \u0026plusmn; 0.26 Log CFU/ml and 10.44 \u0026plusmn; 0.13 Log CFU/ml, n=6, respectively). However, the live bacterial counts in blood and peritoneal lavage in both septic and diabeto-septic mice were found to be significantly higher in comparison to SO and diabetic mice. No bacterial count was detected in blood and peritoneal lavage from SO and diabetic mice. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiv) Effect on\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehigh K\u003csup\u003e+\u003c/sup\u003e (80 mM)-depolarizing solution-induced contraction:\u0026nbsp;\u003c/strong\u003eAs shown in the Fig.4A, sepsis (13 h post-CLP) significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) decreased the maximum aortic response to high K\u003csup\u003e+\u003c/sup\u003e-depolarizing solution (0.28 \u0026plusmn; 0.02 g; n=10) as compared to that observed in SO mice (0.51 \u0026plusmn; 0.01 g; n=10). However, unlike in sepsis, interestingly the maximal contractile response to high K\u003csup\u003e+\u003c/sup\u003e in the aortic rings from mice of the diabeto-septic group (0.46 \u0026plusmn; 0.01 g, n=10) was found to be significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) higher and almost similar to that observed in mice of the SO group. Moreover, this value was significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) lower in comparison to diabetic group (0.65 \u0026plusmn; 0.02 g; n=10). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ev) Effect on NA-induced contraction:\u0026nbsp;\u003c/strong\u003eFig.4B summarizes the aortic reactivity to NA in the mice of different groups (SO, sepsis, diabetes and diabeto-sepsis). \u0026nbsp;The E\u003csub\u003emax\u003c/sub\u003e and pD\u003csub\u003e2\u003c/sub\u003e values of NA in mice of the SO group were\u0026nbsp;0.51 \u0026plusmn; 0.02 g and 7.45 \u0026plusmn; 0.05 (n=8), respectively. However, in comparison to SO mice, the aortic response to NA was found to be significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) reduced (E\u003csub\u003emax\u003c/sub\u003e: 0.28\u0026nbsp;\u0026plusmn; 0.03 g; n=6; Fig. 4B) in mice of the sepsis group (13 h post-CLP) without any change in the potency of the agonist (pD\u003csub\u003e2\u003c/sub\u003e: 7.32\u0026nbsp;\u0026plusmn; 0.08 g; n=6). Interestingly, the concentration response curve of NA in diabeto-septic group was superimposed over the DRC of NA in mice of the SO group whereas the DRC of NA in diabetic group was significantly shifted towards left. The E\u003csub\u003emax\u003c/sub\u003e and pD\u003csub\u003e2\u003c/sub\u003e values of NA in diabeto-septic group were\u0026nbsp;0.43 \u0026plusmn; 0.01 g and 7.41 \u0026plusmn; 0.08 (n=8), respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003evi) Effect on mRNA expression of \u0026alpha;\u003csub\u003e1D\u003c/sub\u003e-adrenoceptor in mouse aorta:\u0026nbsp;\u003c/strong\u003eTo correlate the changes in aortic reactivity to NA in the mice of different groups, mRNA expression of \u0026alpha;\u003csub\u003e1D\u003c/sub\u003e was evaluated in the aorta of the mice of all these three groups. As illustrated in Fig.4C, sepsis significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) attenuated (0.43 \u0026plusmn; 0.08 fold; n=4) while diabetes significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) increased (6.57 \u0026plusmn; 0.05 fold; n=4) the aortic mRNA expression of \u0026alpha;\u003csub\u003e1D\u003c/sub\u003e receptors as compared to that observed in SO group (1.00 \u0026plusmn; 0.00; n=4). However, when sepsis was induced in the mice with pre-existing diabetes, the mRNA expression of \u0026alpha;\u003csub\u003e1D\u003c/sub\u003e in the aorta (1.37 \u0026plusmn; 0.21 fold; n=4) was found to be almost restored to the level as observed in SO group. In addition, the \u0026alpha;\u003csub\u003e1D\u003c/sub\u003e mRNA level in diabeto-septic mice was significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) higher in comparison to that observed in mice of the sepsis alone group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003evii) Effect on acetylcholine-induced vasorelaxation:\u0026nbsp;\u003c/strong\u003eCompared to the maximal relaxant effect of ACh in mice of the SO group (94.58 \u0026plusmn; 1.68 %; n=6), the maximum relaxant response to ACh was significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) reduced in aorta of the septic mouse (39.48 \u0026plusmn; 2.20 %; n=6). However, as shown in Fig.4D, ACh-induced relaxation in aortic rings isolated from the septic mice with pre-existing diabetes was found to be significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) greater (65.78 \u0026plusmn; 3.61 %; n=6) in comparison to that observed in the sepsis alone group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eviii) Histopathological Studies\u003c/strong\u003e\u003c/p\u003e\n\u003col class=\"decimal_type\" start=\"1\" style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003e\u003cstrong\u003ePancreas:\u0026nbsp;\u003c/strong\u003eThe tissue section of pancreas from SO (Fig. 5A) groups showed normal islet of Langerhans having both \u003cem\u003ebeta\u0026nbsp;\u003c/em\u003eand \u003cem\u003ealpha\u003c/em\u003e cells. In chromium-hematoxylin-phloxine staining, \u003cem\u003ebeta\u003c/em\u003e cells take blue colour while \u003cem\u003ealpha\u003c/em\u003e cells appear red. In diabetes groups (Fig. 5B), destruction of \u003cem\u003ebeta\u003c/em\u003e cells with vasculitis was observed, while \u003cem\u003ealpha\u003c/em\u003e cells remain intact. In sepsis group (Fig. 5C), islet of Langerhans was found to be intact but degeneration and congestion of exocrine and acini of pancreas were observed. Further, the acini became coalesce to each other. \u0026nbsp;In diabeto-sepsis group (Fig. 5D), destruction of \u003cem\u003ebeta\u003c/em\u003e cells as well as degeneration and coalesce of exocrine acini along with accumulation of edematous fluid and fibres were observed.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLiver:\u0026nbsp;\u003c/strong\u003eCompared to\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003enormal histo-architecture of liver with central vein and radiating hepatic cords in SO (Fig. 6A) group, the tissue sections from diabetic group (Fig. 6B) showed congestion of central vein in hepatic parenchyma and swelling and degeneration of hepatocytes in hepatic cord with course nuclear material. In sepsis group (Fig. 6C) and diabeto-sepsis groups (Fig. 6D), liver section showed severe congestion in central vein and other parenchymal blood vessels, degeneration of hepatocytes along with infiltration of inflammatory cells in paracentral area. As summarized in Table 1, the comparative histopathological scoring based on the infiltration of inflammatory cells in liver sections from different groups was diabeto-sepsis\u0026gt;sepsis\u0026gt;diabetes= SO control.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Histopathological scoring of different organs of animals from different groups\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"541\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80221811460259%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.19778188539742%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInflammatory cell infiltration in the organs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLiver\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLung\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKidney\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpleen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80221811460259%\" valign=\"top\"\u003e\n \u003cp\u003eSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80221811460259%\" valign=\"top\"\u003e\n \u003cp\u003eDiabetes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80221811460259%\" valign=\"top\"\u003e\n \u003cp\u003eSepsis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80221811460259%\" valign=\"top\"\u003e\n \u003cp\u003eDiabeto-sepsis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.744916820702404%\" valign=\"top\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.853974121996302%\" valign=\"top\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;Grade 0: - negative, Grade 1 (+): 0\u0026ndash;33% mild positive, Grade 2(++): 33\u0026ndash;66% moderate positive, Grade 3 (+++): 66\u0026ndash;100% severe positive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Lungs:\u0026nbsp;\u003c/strong\u003eTissue section of lungs from SO group showed normal architecture of alveoli along with normal alveolar septa (Fig. 7A). In diabetes group (Fig. 7B) constriction of alveoli and thickening of alveolar walls was evident while in septic group (Fig. 7C) lung \u0026nbsp; histopathology was characterized by presence of severe edema, constriction of alveoli and thickening of alveolar septa with infiltration of inflammatory cells. Lung tissue sections from diabeto-sepsis group showed constriction of alveoli and thickening of alveolar septa with congestion of alveolar capillaries and infiltration of cells (Fig. 7D). The histopathological scoring of lung tissue sections from different groups was diabeto-sepsis\u0026gt;sepsis\u0026gt;diabetes\u0026gt; SO control (Table 1).\u003c/p\u003e\n\u003col class=\"decimal_type\" start=\"4\" style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003e\u003cstrong\u003eKidney:\u003c/strong\u003e In diabetes group (Fig. 8B) and sepsis group (Fig. 8C) degeneration of tubules and glomeruli, congestion in renal blood vessels with infiltration of inflammatory cells were prominent. In diabeto-sepsis group (Fig. 8D), congestion and haemorrhages with degeneration and necrosis of tubules and glomeruli, infiltration of inflammatory cells around glomeruli and tubules were comparatively higher. The order of infiltration of inflammatory cells in kidney tissue section was diabeto-sepsis\u0026gt;sepsis\u0026gt;diabetes=SO control (Table 1).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSpleen:\u0026nbsp;\u003c/strong\u003eCompared to the normal orientation of red pulp and white pulp with capsule and trabeculae in SO group (Fig.9A), the tissue sections of spleen from diabetic and sepsis groups showed depletion of white pulp with necrosis of blood channels, deposition of hemosiderin pigements (Fig. 9B \u0026amp; C, respectively). In diabeto-sepsis group, severe congestion of sinusoids and depletion of white pulp at capsular area with excess deposition of hemosiderin pigments was observed (Fig. 9D).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe histopathological scoring based on infiltration of inflammatory cells was diabeto-sepsis\u0026gt;sepsis\u0026gt;diabetes\u0026gt; SO control (Table 1).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Discussion","content":"\u003cp\u003eMajor findings in the present study were i) septic mice with pre-existing diabetes died earlier in comparison to sepsis alone mice ii) vascular reactivity to NA was significantly increased in diabetic mice while decreased in septic mice as compared to control group, however, in diabeto-sepsis group NA-induced contraction was found to be similar to that observed in SO group iii) the mRNA expression of α\u003csub\u003e1D\u0026minus;\u003c/sub\u003eadrenoreceptor in aorta of diabeto-septic mice was also found to be similar to that of SO mice, however, significant increase and decrease in the receptor expression were observed in diabetes and sepsis group, respectively iv) septic mice with pre-existing diabetes also exhibited marked hypoglycemia before death with reduced bacterial load and increased inflammatory cells infiltration in lungs, liver, kidney and spleen in comparison to the mice of diabetes and/or sepsis alone groups.\u003c/p\u003e \u003cp\u003ePatho-physiology of sepsis in diabetes has been studied in number of animal models and most of these studies have primarily focused on immune system. However, impact of sepsis in pre-existing diabetes on cardio-vascular system has not attracted much attention of the scientific community. Dysregulation of cytokines functions and prolonged inflammatory response is considered to be the underlying cause of higher mortality in diabetic mice subjected to Group B \u003cem\u003eSterptococci\u003c/em\u003e-induced sepsis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In spontaneous type-1 diabetes (T1D) as well as Goto-Kakizaki T2D model, the mortality rate in animals was enhanced following caecal ligation and puncture (CLP)-induced sepsis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In addition, alloxan-induced T1D mouse was found to be highly susceptible to polymicrobial sepsis due to G-protein-coupled receptor kinase-2 (GRK2)-mediated down-regulation of chemokine receptor (CXCR-2) on blood neutrophils resulting in aberrant neutrophil migration [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In agreement to these observations, we also observed a significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in survival time in the mice of diabeto-sepsis group compared to the sepsis alone group in the present study.\u003c/p\u003e \u003cp\u003eIn contrast to the existing report about early mortality in complicated sepsis (sepsis in pre-existing diabetes), some research findings and epidemiological data suggest less likelihood about development of acute lung injury in septic patients with the history of diabetes as compared to the septic-patients without diabetes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] possibly due to reduced activation of NF-κB in alveolar macrophage and decreased expression of MyD88 in lungs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, this reduced inflammatory response in diabetic rats subjected to sepsis seems to be more restricted to lungs since diabetic patients are more likely to show enhanced renal dysfunction following development of sepsis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus, the impact of pre-existing diabetes on sepsis-induced multiple organ dysfunctions is a complex process and seems to be organ specific. In the present study, we aimed to compare the effect of pre-existing diabetes and sepsis with that of sepsis alone in respect of vascular function/reactivity. Circulatory failure in sepsis is characterized by refractory hypotension and vascular hyporeactivity to clinically used vasoconstrictors like nor-adrenaline which leads to impaired tissue perfusion and multi-organ dysfunction. Overproduction of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) along with down-regulation and desensitization of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptor expression are considered to be the underlying mechanism of vascular hyporeactivity in sepsis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Thus, we aimed to study the aortic response to NA, a commonly used vasopressor agent, in diabeto-septic mice. To our surprise, we did not observe any hyporeactivity to NA in the aortic rings from mice of the diabeto-septic group as observed in septic mice. Our observation of functional study (isometric tension measurement) is also strongly corroborated with the mRNA expression of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptors in aortic rings where this receptor expression was found to be statistically similar in both diabeto-sepsis and SO groups. However, in consistent with the aortic reactivity to NA, mRNA expression of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptors was down-regulated in sepsis alone group whereas up-regulated in diabetes group. Earlier report from our laboratory [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] has also demonstrated the down-regulation and desensitization of α\u003csub\u003e1D\u0026minus;\u003c/sub\u003eadrenoceptors in aorta following polymicrobial sepsis in mice. But on the contrary, increase in α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptors was reported in diabetes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Thus, the vascular reactivity to NA and expression of mRNA transcript of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptor in aorta of the diabeto-septic mice in our study indicate that when diabetic mice were subjected to polymicrobial sepsis, sepsis-induced vascular hyporeactivity to NA and associated down-regulation of α\u003csub\u003e1D\u003c/sub\u003e expression in aorta were counteracted by diabetic state of the animals. Further, similar to the aortic reactivity to NA, the maximum contractile response to high K\u003csup\u003e+\u003c/sup\u003e (indicative of voltage-gated Ca\u003csup\u003e2+\u003c/sup\u003e channel-dependent contraction) in diabeto-sepsis group was also found to be almost comparable to that observed in mice of SO group, while it was found to be significantly decreased in mice of the sepsis group and significantly increased in the aortic rings of diabetes group. Thus apparently, the receptor-independent and voltage-gated Ca\u003csup\u003e2+\u003c/sup\u003e channel-dependent arterial contractile response also remained unaltered in septic mice with pre-existing diabetes. Therefore, suggesting that diabeto-sepsis subjects are not prone to hypotensive crisis as in sepsis or hypertensive crisis as in diabetes.\u003c/p\u003e \u003cp\u003eIt has been documented that during septic shock, patients with diabetes have higher circulating levels of inflammatory biomarkers like endothelial cell adhesion (E-selectin) and vascular endothelial growth factor (sFLT-1) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, compared to sepsis alone, we did not observe any augmentation in endothelial dysfunction in the aortic rings from diabeto-septic mice as there was no significant alteration in the vasorelaxant response to ACh in the septic mice with pre-existing diabetes as compared to that in sepsis group. Thus, ruling out the possible involvement of impaired vasorelaxant response in sepsis-induced reduction in survival time in pre-existing diabetic mice.\u003c/p\u003e \u003cp\u003eHypoglycemia results in poor outcome in diabetic patients [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. On the other hand, septic patients are often encountered with stress-induced hyperglycemia [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], however, the severity of hyperglycemia and nature of critical illness determine the prognosis in septic patients [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Hyperglycemia in sepsis is not only a marker of severity but also governs the adverse outcome in different vital organs during sepsis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It disrupts the functions of innate immunity by reducing neutrophil chemotaxis, formation of reactive oxygen species and phagocytosis of bacteria despite accelerated infiltration of leukocytes into the peripheral tissues [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. But numerous studies have reported an association between hypoglycemia and high mortality in severely ill septic patients [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The hypoglycemic state in sepsis is thought to be due to cytokine-mediated reduction in glucose production [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and down-regulation of GLUT2 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Hypoglycemia in sepsis may develop despite increased glucose production and is mediated by non-insulin dependent increased uptake and utilization of glucose in tissues rich in macrophages like liver, lung, spleen and ileum [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Consistent with these reports, we also observed an early hyperglycemic stage followed by euglycemia and hypoglycemia in septic mice whereas early hyperglycaemia followed by hypoglycaemic phase in diabeto-septic mice. On histopathological examination, marked infiltration of inflammatory cells was observed in macrophage-rich tissues like liver, lung, kidney and spleen of the mice of diabeto-sepsis group. Apart from this, significant decrease in total bacterial counts was also observed in the peritoneal lavage (source of infection) and blood (systemic infection) of the mice of diabeto-sepsis group. Thus, suggesting that heavy infiltration of inflammatory cells in different tissues reduces the bacterial load in this group of mice and is responsible, at least in part, for reduction in blood glucose level leading to hypoglycemic shock in diabeto-septic subjects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTaken together, based on the findings of present study, it may be inferred that the early mortality in septic mice with pre-existing diabetes (in comparison to sepsis alone) possibly seems to be due to increased infiltration of inflammatory cells and associated hypoglycemia (non-insulin dependent) rather than vascular dysfunction as observed in sepsis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The research work is not supported by any grants from specific Funding Agency. However, financial assistance under the \u0026ldquo;Strengthening and Development Grant to the University\u0026rdquo; by the Indian Council of Agricultural Research, New Delhi, for support of routine research work of postgraduate students is thankfully acknowledged. The laboratory facility established by the Department of Veterinary Pharmacology and Toxicology, DUVASU, Mathura, India, under ICAR-sponsored Niche Area of Excellence Programme (Grant No. 10 (10)/2012-EPD dated 23rd march 2012) is also thankful acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u0026nbsp; \u0026nbsp;None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003eMG conducted the\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efunctional study, MG, TJ and SC conducted the mRNA expression study, MG, AS, NG performed the histopathological experiments, AS, SC, MG analyzed the data, SC and SKG conceptualized the experiments and wrote the MS. All the authors have read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBosmann, M., \u0026amp; Ward, P. A. (2013). The inflammatory response in sepsis. \u003cem\u003eTrends in immunology\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e(3), 129\u0026ndash;136. https://doi.org/10.1016/j.it.2012.09.004\u003c/li\u003e\n\u003cli\u003eAngus, D. C., Linde-Zwirble, W. T., Lidicker, J., Clermont, G., Carcillo, J., \u0026amp; Pinsky, M. R. (2001). Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. \u003cem\u003eCritical care medicine\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e(7), 1303\u0026ndash;1310. https://doi.org/10.1097/00003246-200107000-00002\u003c/li\u003e\n\u003cli\u003eKochanek, K. D., \u0026amp; Smith, B. L. (2004). 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Alterations in tissue glucose uptake during the hyperglycemic and hypoglycemic phases of sepsis. \u003cem\u003eShock (Augusta, Ga.)\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(5), 379\u0026ndash;385. https://doi.org/10.1097/00024382-200005000-00006\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":"sepsis, diabetes, hypoglycemia, vascular dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-3857212/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3857212/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepsis is often complicated with pre-existing diabetes and diabetic patients are prone to infection. However, the impact of sepsis in pre-existing diabetes especially on cardio-vascular system is largely unknown. Sepsis was induced by caecal ligation and puncture while intra-peritoneal injection of streptozotocin (@ 65 mg/kg b.wt. for 5 consecutive days) was used to induce diabetes in mice. Isometric tension and mRNA expression of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptor in aorta was determined by organ bath and qRT-PCR, respectively. Blood glucose levels and bacterial load in blood and peritoneal lavage (PL) were estimated. Histopathological examination of pancreas, lungs, liver, kidney and spleen was also done. Induction of sepsis in the mice with pre-existing diabetes caused early mortality despite being lower bacterial load in blood and PL in comparison to sepsis alone. Interestingly, NA-induced contraction as well as receptor-independent high K\u003csup\u003e+\u003c/sup\u003e-induced contraction (though significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduced in sepsis), were similar in diabeto-septic and SO groups. Accordingly, aortic mRNA expression of α\u003csub\u003e1D\u003c/sub\u003e-adrenoceptor was also unaltered in diabeto-septic group unlike to that of sepsis where α\u003csub\u003e1D\u003c/sub\u003e mRNA expression was significantly down-regulated. ACh-induced vasorelaxation was also unaffected in these animals. However, marked hypoglycemia before death with enhanced infiltration of inflammatory cells in lungs, liver, kidney and spleen was observed. In diabeto-septic animals, hypoglycaemia rather than vascular dysfunction was responsible for early mortality. Further, the increased infiltration of inflammatory cell in different tissues reduced the bacterial load and is responsible, at least in part, for reduction in blood glucose level leading to hypoglycemic shock.\u003c/p\u003e","manuscriptTitle":"Hypoglycemia rather than vascular dysfunction causes early mortality in diabeto-septic mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 20:36:58","doi":"10.21203/rs.3.rs-3857212/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":"c98db856-7f69-4d9c-807d-9a8582312b33","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-12T20:31:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-16 20:36:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3857212","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3857212","identity":"rs-3857212","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}