Hindlimb Function Research Articles

Late acquisition of erect hindlimb posture and function in the forerunners of therian mammals.

The evolutionary transition from early synapsids to therian mammals involved profound reorganization in locomotor anatomy and function, centered around a shift from "sprawled" to "erect" limb postures. When and how this functional shift was accomplished has remained difficult to decipher from the fossil record alone. Through biomechanical modeling of hindlimb force-generating performance in eight exemplar fossil synapsids, we demonstrate that the erect locomotor regime typifying modern therians did not evolve until just before crown Theria. Modeling also identifies a transient phase of increased performance in therapsids and early cynodonts, before crown mammals. Further, quantifying the global actions of major hip muscle groups indicates a protracted juxtaposition of functional redeployment and conservatism, highlighting the intricate interplay between anatomical reorganization and function across postural transitions. We infer a complex history of synapsid locomotor evolution and suggest that major evolutionary transitions between contrasting locomotor behaviors may follow highly nonlinear trajectories.

Effectiveness of Insolubilized Poly(vinyl alcohol)-Based Electrospun Fiber-Loaded Methylprednisolone by Enzyme-Catalyzed Cross-Linking in a Rat Spinal Cord Injury Model.

Spinal cord injury (SCI) has been implicated in neural loss and, consequently, motor/sensory impairment. Here, we propose an improved formation for fibrous mat fabrication from the derivatives of poly(vinyl alcohol) (PVA) and gelatin (Gela) through horseradish peroxidase-mediated cross-linking, providing a sustained release of methylprednisolone (MP) for SCI repair. After 28 days, the animals were evaluated in terms of remyelination and apoptosis and underwent behavioral tests. The mechanical properties, hydrophobicity, and degradation rate of PVAPh/GelaPh fibrous mats were significantly improved as compared with those of PVAPh samples. This could provide the desired structure for a sustained MP release. The seeded cells could adhere and proliferate to the composite fibers, which indicates the cytocompatibility of the resultant PVAPh/GelaPh fibrous mat. The results showed significant reductions in the number of apoptotic neurons and a substantial improvement in remyelination in the SCI+ PVAPh/GelaPh + MP group. The behavioral tests confirmed improvement in locomotor hindlimb function following treatment. The MP-loaded PVAPh/GelaPh mat developed through the long-term release of MP and the biocompatible fabricated mat could inhibit axonal demyelination, attenuate apoptosis, and improve the functional outcome, which verified the potential of PVAPh/GelaPh + MP nanocomposites as a bioactive scaffold for SCI regeneration.

The NCX1 calcium exchanger is implicated in delayed axotomy after peripheral nerve stretch injury.

After peripheral nerve stretch injury, most degenerating axons are thought to become disconnected at the time of injury, referred to as primary axotomy. The possibility of secondary axotomy-a delayed and potentially reversible form of disconnection-has not been evaluated. Here, we investigated secondary axotomy in a rat model of sciatic nerve stretch injury. We also evaluated whether axon sparing and functional improvement results from pharmacological blockade of the sodium-calcium exchanger 1 (NCX1), which is widely believed to contribute to traumatic axon degeneration but was previously only investigated in vitro. We studied peripheral nerve secondary axotomy in a clinically relevant rat model of sciatic nerve rapid stretch injury with immunolabeling and fluorescence microscopy. The role of NCX1 in secondary axotomy was studied with pharmacological inhibition with SEA0400 and immunolabeling, immunoblot, and behavioral assays. We found that early after injury, many axons remained in-continuity and that degeneration of axons was delayed, consistent with the occurrence of secondary axotomy. βAPP, a marker of secondary axotomy, accumulated at regions of axon swelling and disconnection, and NCX1 was upregulated and co-localized to βAPP axonal swellings. Pharmacological blockade of NCX1 after injury reduced calpain activation, proteolytic degradation of neurofilaments, βAPP accumulation, distal axon degeneration, and improved hindlimb function. Our data demonstrate a major role for secondary axotomy in peripheral nerve stretch injury and identify NCX1 as a promising therapeutic target to reduce secondary axotomy and improve functional outcome after nerve injury.

Development of a chronic compression spinal cord injury model in neonatal and adult rats.

Spinal cord injury presents a significant burden globally, with traumatic spinal cord injury being the predominant cause historically. However, nontraumatic spinal cord injury (NTSCI) is emerging as a significant contributor, particularly in developed nations, yet it remains poorly understood due to underreporting and misclassification. NTSCI, spanning various etiologies such as bony growths, vascular conditions, infections, neoplastic conditions, and immune disorders, poses unique challenges in diagnosis and treatment, often resulting in lifelong morbidity. This study addresses the lack of suitable animal models for NTSCI research, especially in neonatal animals. Utilizing a solid spacer approach, we developed a compression NTSCI model applicable to both neonatal and adult Sprague-Dawley rats. Through anatomical measurements and invivo experiments, we confirmed the feasibility and safety of the spacer insertion procedure and observed no acute off-target effects. The versatility of this model lies in its adaptability to different ages of rats, offering a cost-effective and reproducible means to induce graded injuries. Moreover, behavioral assessments demonstrated observable hindlimb function, validating the model's utility for studying functional outcomes. Although challenges persist, particularly in accounting for spinal column growth in neonatal animals, this model fills a crucial gap in pediatric NTSCI research. By providing a platform to investigate pathophysiological mechanisms and test potential treatments, it offers promising avenues for advancing our understanding and management of pediatric NTSCI.

NeuroAiDTM-II (MLC901) Promoted Neurogenesis by Activating the PI3K/AKT/GSK-3β Signaling Pathway in Rat Spinal Cord Injury Models.

Traumatic damage to the spinal cord (SCI) frequently leads to irreversible neurological deficits, which may be related to apoptotic neurodegeneration in nerve tissue. The MLC901 treatment possesses neuroprotective and neuroregenerative activity. This study aimed to explore the regenerative potential of MLC901 and the molecular mechanisms promoting neurogenesis and functional recovery after SCI in rats. A calibrated forceps compression injury for 15 s was used to induce SCI in rats, followed by an examination of the impacts of MLC901 on functional recovery. The Basso, Beattie, and Bresnahan (BBB) scores were utilized to assess neuronal functional recovery; H&E and immunohistochemistry (IHC) staining were also used to observe pathological changes in the lesion area. Somatosensory Evoked Potentials (SEPs) were measured using the Nicolet® Viking Quest™ apparatus. Additionally, we employed the Western blot assay to identify PI3K/AKT/GSK-3β pathway-related proteins and to assess the levels of GAP-43 and GFAP through immunohistochemistry staining. The study findings revealed that MLC901 improved hind-limb motor function recovery, alleviating the pathological damage induced by SCI. Moreover, MLC901 significantly enhanced locomotor activity, SEPs waveform, latency, amplitude, and nerve conduction velocity. The treatment also promoted GAP-43 expression and reduced reactive astrocytes (GFAP). MLC901 treatment activated p-AKT reduced p-GSK-3β expression levels and showed a normalized ratio (fold changes) relative to β-tubulin. Specifically, p-AKT exhibited a 4-fold increase, while p-GSK-3β showed a 2-fold decrease in T rats compared to UT rats. In conclusion, these results suggest that the treatment mitigates pathological tissue damage and effectively improves neural functional recovery following SCI, primarily by alleviating apoptosis and promoting neurogenesis. The underlying molecular mechanism of this treatment mainly involves the activation of the PI3K/AKT/GSK-3β pathway.

AB015. Efficacy and safety of boron neutron capture therapy in managing metastatic spinal tumors: experimental findings.

Boron neutron capture therapy (BNCT) is a unique cancer treatment modality that enables precise targeting of tumors at the cellular level. Based on the success observed in nuclear reactors, BNCT now holds promise as a therapeutic approach for treating invasive brain tumors or head and neck cancers. Metastatic spinal tumors have been treated with multidisciplinary interventions such as surgical resection and radiation therapy. Despite recent advantages of radiation therapy, it remains challenging to achieve better quality of life and activity of daily living. The purpose of this study was to evaluate the efficacy and safety of BNCT in metastatic spinal tumor using a mouse model. For the in vitro, neutron and photon irradiation was applied to A549 human lung adenocarcinoma cells. The cells were irradiated neutrons with or without p-boronophenylalanine (BPA) 10 µg Boron/mL for a 24-h exposure before neutron irradiation. The difference of biological effect between neutrons and photons was evaluated by colony forming assay. For in vivo, the tumor-bearing mice were intravenously administered BPA (250 mg/kg), followed by measuring biodistribution of boron using inductively coupled plasma atomic emission spectroscopy (ICP-AES). For in vivo BNCT, the mice were randomly assigned to untreated (n=10), neutron irradiation only (n=9), and BNCT groups (n=10). Overall survival and hindlimb function were analyzed. Histopathological examination was also performed to assess the influences of neutron irradiation. Neutron irradiation showed a stronger cell-killing effect than that exhibited by photon irradiation in vitro. For in vivo biodistribution, the highest boron accumulation in the tumor was seen at 2.5-h time point (10.5 µg B/g), with a tumor to normal spinal cord and blood ratios were 3.6 and 2.9, respectively. For the in vivo BNCT, BNCT had significantly prolonged survival (vs. untreated, P=0.002; vs. neutron only, P=0.01, respectively, log-rank test) and preserved mice hindlimb function compared to the other groups (vs. untreated, P<0.001; vs. neutron only, P=0.005, respectively, MANOVA). No adverse events and apparent histopathological changes were observed among three groups. These findings indicate that BNCT may represent a novel therapeutic option in the management of metastatic spinal tumors.

Mouse developmental defects, but not paraganglioma tumorigenesis, upon conditional Complex II loss in early Sox10+ cells.

In humans, loss of heterozygosity for defective alleles of any of the four subunits of mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH, also Complex II of the electron transport chain) can lead to paraganglioma tumors in neuroendocrine cells. With the goal of developing mouse models of this rare disorder, we have developed various SDH conditional loss strategies. Based on recent lineage tracing studies, we hypothesized that conditional SDHC loss in early embryogenesis during migration of primordial neural crest cells that form the susceptible chromaffin cells of the adrenal medulla might induce paraganglioma. We triggered low levels of detectable SDHC loss in Sox10+ cells at E11.5 of mouse development. We report that, rather than developing adrenal medulla paraganglioma (pheochromocytoma), offspring survived with evidence of neural crest cell dysfunction. Phenotypes included mild lower extremity gait anomalies suggestive of neural tube closure defects and patches of unpigmented fur consistent with neural crest-derived melanocyte dysfunction. These defects were not observed in mice lacking Sdhc knockout. Our results add to existing data suggesting that, unlike humans, even early embryonic (Sox10-driven) SDHx loss is inadequate to trigger paraganglioma in mice of the genetic backgrounds that have been investigated. Instead, low levels of tricarboxylic acid cycle-deficient neural crest cells cause mild developmental defects in hind limb and melanocyte function. This new model may be of interest for studies of metabolism during early neural crest cell development.

Implantation of Optoelectronic Devices in the Rodent Spinal Cord.

Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the spinal cord remains relatively unexplored. The inherently delicate and mobile spinal cord tissue imposes constraints that make the precise implantation of neural probes challenging. Despite recent advances in neuromodulation devices, particularly flexible bioelectronics, opportunities to expand their use in the spinal cord have been limited by the surgical complexities of device implantation. Here, we provide a series of surgical protocols tailored specifically for the implantation of a custom-made optoelectronic device that interfaces with the spinal cord in rodents. The steps to place and anchor an optical shank on a specific segment of the spinal cord via two different surgical implantation methods are detailed here. These methods are optimized for a diverse range of devices and applications, which may or may not require direct contact with the spinal cord for optical stimulation. To elucidate the methodology, the vertebral anatomy is referenced first to identify prominent landmarks before making a skin incision. The surgical steps to secure an optical shank over the cervical spine in rodents are demonstrated. Procedures are then outlined for securing the optoelectronic device connected to the optical shank in a subcutaneous space away from the spinal cord, minimizing unnecessary direct contact. Behavioral studies comparing animals receiving the implants to those undergoing sham surgeries indicate that the optical shanks did not adversely affect hindlimb or forelimb function seven days post-implantation. The present work broadens the neuromodulation toolkit for use in future studies aimed at investigating various spinal cord interventions.

Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug enabling of subsequent clinical studies. Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia. Understanding the dose for effective gene delivery is crucial for future Investigational New Drug-enabling studies. Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved 3 cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly in divided aliquots, ranging from 2 × 10 9 VG to 2 × 10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery, and therapeutic angiogenesis were assessed. Codon-optimized E-Sel/AAV2 gene therapy exhibits a superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2 × 10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber scores, increased running stamina, and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. E-Sel/AAV2 Vascular Regeneration Gene Therapy holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2 × 10 11 VG aliquots injected intramuscularly.

Biosynthesis of gold nanoparticles by using gelatin as reducing and stabilizing agent under ultrasound irradiation: Application of its catalytic activity and for ameliorating motor recovery after spinal cord injury in the Wistar rat’s model

The current project describes the facile preparation of Au nanoparticles mediated by gelatin as a natural capping and reducing agent in mild condition. The formation of Au nanoparticles stabilized by gelatin (Au NPs/Gelatin) and structural characteristics were studied via UV–Vis, FT-IR, TEM, FE-SEM, EDX and elemental mapping techniques. The evidence shown that gelatin-capped gold nanoparticles resulted in the lowest average particle sizes of 15–25 nm and spherical shapes nanoparticles. Further, newly synthesized Au NPs/Gelatin are used as a nanocatalyst for the reduction of 4-nitrophenol (4-NP) dyes. A UV–Vis spectrophotometer is employed to track the effectiveness of the reduction process. Recyclability in six further degradation cycles is used to assess the sustainability of this catalyst, and the results show a steady and notable degradation activity. In the in vivo experiment, 60 male rats were classified into four groups: control, intact, sham, and Au NPs/Gelatin nanocatalyst (20 µg/kg) groups. The Hematoxylin and eosin staining was conducted to examine the post-injury lesions. Additionally, somatosensory evoked potential experiments were conducted to assess the recovery of neural conduction. The expression of GFAP was evaluated to determine the extent of astrogliosis. Furthermore, the behavioral outcomes of the rats were evaluated using the BBB scores on a weekly basis after the spinal cord injury onset. The results of the research demonstrated that the neuroprotective effects of Au NPs/Gelatin nanocatalyst led to an amelioration in the rat’s model. Electromyography (EMG) data revealed a remarkable amelioration in hindlimb function in the Au NPs/Gelatin nanocatalyst (20 µg/kg) group. The ventral motor neurons number significantly raised, while the cavity areas significantly decreased in the Au NPs/Gelatin group. Moreover, the expression of GFAP was notably reduced in the Au NPs/Gelatin nanocatalyst group. The Au NPs/Gelatin nanocatalyst group exhibited a significant reduction in delayed responses and a significant increase in BBB scores during sensory tests.

Abstract 3032: Novel GLP-Grade E-Selectin/AAV2 Gene Therapy Optimal Dosage For Vascular Regeneration

Introduction: Chronic Limb Threatening Ischemia (CLTI) represents a major public health burden with with an estimated 15 to 20% CLTI patients undergoing major limb amputation facing a 50% mortality risk at 5 years post amputation. We previously demonstrated the pro-angiogenic actions of a research-grade E-Selectin/AAV gene therapy in a hindlimb gangrene murine model. In this study, a dosage study utilizing GLP grade codon-optimized and DeNovo-synthesized E-selectin/AAV2 was conducted dosage optimization and determination of the necessary scale-up for subsequent clinical trials. Methods: codon-optimized and de novo synthesized human and murine E-selectin AAV vectors under GLP conditions was evaluated in vitro by cell transduction assays. E-selectin/AAV2 i.m. injection in three cohorts of FVB/NJ mice (9-12 weeks old, N=6/group) with GFP/AAV2 (9-12 weeks old, N=6/group) groups, was performed in vivo in escalating log doses ranging from 2x10^9 VG to 2x10^11 VG. Hindlimb ischemia and gangrene were subsequently induced through left femoral artery/vein ligation/excision (FAVLE). Limb perfusion recovery and function was assessed postoperatively by Laser Doppler Imaging (LDI), Faber hindlimb ischemia scores, treadmill exhaustion test, whole-body Dil perfusion Results: Treatment with high dose (2x10^11 VG) of E-selectin/AAV2 significantly improve perfusion indices compared to control groups (p=.0019, N=6/group) and lower doses (p=.0006 and p=.0082, N=6/group) with decreased Faber’s scores. A significant increase in neovascularization was also observed compared to GFP control (0.41 ±0.14 to 0.95±0.18; p=.0080, N=5/group) and lower doses (p=.0026 and p=.009, N=5/group). Running stamina was also significantly improve by VRGT with a 2x10^11 VG dosage treatment compared to GFP (p=0.0047) control and lower doses, establishing a clear dose-dependent response. Conclusion: VGRT improves recovery of ischemic hindlimb perfusion and function at an optimal dose of 2x10 11 VG. This pre-clinical study paves the way towards further studies using the same AAV construct manufactured under cGMP conditions and subsequent Investigational New Drug (IND) process for clinical pilot studies and disease burden relief.

Safety of mapping the motor networks in the spinal cord using penetrating microelectrodes in Yucatan minipigs.

The goal of this study was to assess the safety of mapping spinal cord locomotor networks using penetrating stimulation microelectrodes in Yucatan minipigs (YMPs) as a clinically translational animal model. Eleven YMPs were trained to walk up and down a straight line. Motion capture was performed, and electromyographic (EMG) activity of hindlimb muscles was recorded during overground walking. The YMPs underwent a laminectomy and durotomy to expose the lumbar spinal cord. Using an ultrasound-guided stereotaxic frame, microelectrodes were inserted into the spinal cord in 8 animals. Pial cuts were made to prevent tissue dimpling before microelectrode insertion. Different locations within the lumbar enlargement were electrically stimulated to map the locomotor networks. The remaining 3 YMPs served as sham controls, receiving the laminectomy, durotomy, and pial cuts but not microelectrode insertion. The Porcine Thoracic Injury Behavioral Scale (PTIBS) and hindlimb reflex assessment results were recorded for 4 weeks postoperatively. Overground gait kinematics and hindlimb EMG activity were recorded again at weeks 3 and 4 postoperatively and compared with preoperative measures. The animals were euthanized at the end of week 4, and the lumbar spinal cords were extracted and preserved for immunohistochemical analysis. All YMPs showed transient deficits in hindlimb function postoperatively. Except for 1 YMP in the experimental group, all animals regained normal ambulation and balance (PTIBS score 10) at the end of weeks 3 and 4. One animal in the experimental group showed gait and balance deficits by week 4 (PTIBS score 4). This animal was excluded from the kinematics and EMG analyses. Overground gait kinematic measures and EMG activity showed no significant (p > 0.05) differences between preoperative and postoperative values, and between the experimental and sham groups. Less than 5% of electrode tracks were visible in the tissue analysis of the animals in the experimental group. There was no statistically significant difference in damage caused by pial cuts between the experimental and sham groups. Tissue damage due to the pial cuts was more frequently observed in immunohistochemical analyses than microelectrode tracks. These findings suggest that mapping spinal locomotor networks in porcine models can be performed safely, without lasting damage to the spinal cord.

Heat therapy preconditioning improves the skeletal muscle contractile and mitochondrial function in a mouse model of access-related hand dysfunction

Introduction: Hemodialysis via arteriovenous fistula (AVF) is a life-line treatment for patients with end-stage kidney disease. However, hemodialysis access-related hand dysfunction (ARHD) including ischemia, muscle weakness and alterations in mitochondrial function is prevalent following AVF placement. We tested the hypothesis that two-weeks of daily heat therapy prior to AVF surgery would improve hindlimb function in a mouse model of ARHD. Methods: Adult male C57BL/6J mice (N=9-11/group) were fed a 0.2% adenine diet to induce chronic kidney disease. Following two weeks of the initial adenine diet, animals were randomly allocated to heat therapy (HT) or control (CON) groups. Animals assigned to HT were exposed to environmental heat at 40°C in a climate chamber for 30 min/day, six days/week, for two weeks while the control mice were exposed to room temperature (~22°C). The AVF was created using the left common iliac artery and vein 24-48 hours after the last treatment session. Laser Doppler perfusion to the ventral paw was assessed post-operatively (POD) and AVF patency was verified using a high-frequency duplex ultrasound system three days prior to euthanasia. Following 14 days of recovery, in situ tibialis anterior (TA) muscle contractility and gastrocnemius muscle mitochondrial function were assessed. RNA sequencing analysis of the hindlimb skeletal muscle (N=4/group) was performed. Results: Compared to baseline, the level of blood urea nitrogen was similarly elevated ( P&lt;0.05) in both groups following four weeks of adenine diet. In both groups, blood perfusion to the paw was decreased by ~50% immediately after AVF creation and progressively recovered to ~80% at POD13 without a treatment effect ( P=0.4467). No differences in body weight and muscle mass in the TA, extensor digitorum longus, and soleus muscles were observed (all P&gt;0.05). Notably, however, the absolute ( P=0.0250) and specific force ( P=0.0101) frequency relationship of the TA muscle were significantly improved in the animals exposed to HT. In addition, compared with control animals, HT mice had significant improvement in oxidative phosphorylation (OXPHOS) in both pyruvate ( P=0.0145) and octanoylcarnitine ( P=0.0196) fueled conditions. RNA sequencing results revealed that genes involved in nervous system development and metabolism were significantly different between HT and CON groups. Conclusion: Daily application of heat therapy for two weeks prior to the AVF surgery has beneficial effects on both skeletal muscle contractile and mitochondrial function suggesting this pre-habilitation strategy may mitigate the risk of ARHD in patients undergoing AVF surgery. Funding Support: National Institutes of Health grant R01HL148597 and American Heart Association grant POST903198. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel Method to Assess Treatment Effcacy in Preclinical Peripheral Arterial Disease Studies

Background: Peripheral arterial disease (PAD), a condition caused by atherosclerotic narrowing or occlusion of blood vessels in the lower extremities, affects ~200 million adults globally and lacks effective treatment options to improve limb function. Cilostazol is the only approved pharmacological treatment option for PAD patients, but its effect on walking performance is modest and the drug is not widely used clinically. Over the past several decades, numerous therapies (drug, gene, and cell-based) have shown promise in the preclinical setting but failed to achieve primary outcomes in clinical trials. While the failure of therapeutic translation is complex, most preclinical studies have employed perfusion recovery as a primary outcome variable whereas most clinical trials in PAD patients employ the six-minute walk test. Although perfusion is an important contributor to limb function and walking performance, assessing preclinical therapeutics solely via perfusion recovery may be a flawed approach. Objective: To address the need for a more comprehensive method to assess the effcacy of preclinical PAD therapeutics, we aimed to develop a protocol to assess hindlimb function that incorporates assessment of muscle power and perfusion across a six-minute testing period. Methods: To assess muscle contractile performance, the tibialis anterior or plantarflexor muscles were carefully exposed and the distal tendon mounted to the force-length controller (Aurora Scientific) and the muscles were stimulated via the sciatic nerve in anesthetized C57BL6 mice. After obtaining isometric forces, a six-minute functional test was performed using repeated isotonic (shortening) contractions against a submaximal load (30% or 45% of the reference force). Muscle fatigue was measured as a change in power across the testing period. Throughout the test, laser Doppler flowmetry was used to measure muscle blood flow. Hindlimb ischemia was used as an experimental model of PAD. Results: In healthy mice without PAD, the test produced the expected dose-dependent relationship between fatigue and workload with contractions performed at 30% of the reference force having less fatigue compared to 45% of the reference force ( P=0.0051), without a significant difference in muscle blood flow between the conditions. Compared the mice that received a sham surgery, those subjected to hindlimb ischemia displayed significantly lower maximal isometric forces (1514 ± 118 mN vs. 880 ± 85 mN, P=0.0001) and power at 35% maximal force (280 ± 12 vs. 105 ± 46 watts/kg, P=0.0002). Whereas sham mice lost ~25% power across the six-minute test, HLI mice displayed a ~70% loss of power. Regarding muscle blood flow, sham mice increased blood flow ~250% whereas HLI mice were only able to increase blood flow to ~105% ( P&lt;0.001). Conclusions: This newly developed protocol successfully combines isotonic muscle function and limb perfusion assessments in the hindlimb of mice and can detect differences between healthy and PAD mice. Future work is needed to fully evaluate the accuracy and sensitivity of this method to select therapeutics in preclinical PAD models. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analgesic Mechanism of Dexmedetomidine and Esketamine in Rats with Spinal Cord Injury.

Spinal cord injury (SCI) is usually caused by external direct or indirect factors, and with a high morbidity and mortality rate. The aim of this study was to observe the effects of Dexmedetomidine (DEX) combined with Esketamine (ESK) on pain behavior and potential analgesic mechanisms in rats with SCI. The goal was to provide a reliable multimodal analgesic medication regimen for SCI. Thirty rats were divided into five groups with six rats in each group: Sham group, SCI group, DEX group, ESK group, and DEX+ESK group. The SCI model in rats was constructed, and the motor function of hind limbs of rats was measured using Basso Beattie Bresnahan (BBB) locomotor rating scale and inclined plate test. The levels of interleukin 18 (IL-18), interleukin 1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the spinal cord were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of substance P (SP), neurokinin-1 receptor (NK-1R), B cell lymphoma-2 (Bcl-2), and Bcl2-associated X protein (Bax) in the rats' spinal cord were measured by Western blot assay. The viability of spinal astrocytes was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 7 days, the BBB scores were significantly higher in the DEX, ESK, and DEX+ESK groups compared to the SCI group (p < 0.01). Additionally, the DEX+ESK group had significantly higher scores than both the DEX and ESK groups (p < 0.01). The maximum angle of the DEX (p < 0.05), ESK (p < 0.05), and DEX+ESK groups (p < 0.01) were higher than the SCI group, and the maximum angle of DEX+ESK group was higher than DEX and ESK groups (p < 0.05). The levels of IL-18, IL-1β, and TNF-α in the DEX, ESK, and DEX+ESK groups were lower than the SCI group (p < 0.01), while the DEX+ESK group had significantly lower IL-18, IL-1β, and TNF-α levels than the DEX and ESK groups (p < 0.01). The levels of SP (p < 0.01) and NK-1R (p < 0.05) were lower in the DEX, ESK, and DEX+ESK groups compared to the SCI group, and the levels of SP and NK-1R were lower in the DEX+ESK group compared to the DEX and ESK groups (p < 0.01). The DEX and ESK groups suppressed the activity of spinal astrocytes (p < 0.01), however, the DEX+ESK group had larger effects on spinal astrocytes than the ESK group (p < 0.05). Treatment using DEX combined with ESK improves the motor function, inhibits inflammation and astrocyte activity, and exerts analgesic effects on rats with SCI. These findings can serve as a reference for the selection of multi-modal analgesics.

Chronic Spinal Cord Injury Regeneration with Combined Therapy Comprising Neural Stem/Progenitor Cell Transplantation, Rehabilitation, and Semaphorin 3A Inhibitor.

Spinal cord injury (SCI) often results in various long-term sequelae, and chronically injured spinal cords exhibit a refractory feature, showing a limited response to cell transplantation therapies. To our knowledge, no preclinical studies have reported a treatment approach with results surpassing those of treatment comprising rehabilitation alone. In this study of rats with SCI, we propose a novel combined therapy involving a semaphorin 3A inhibitor (Sema3Ai), which enhances axonal regeneration, as the third treatment element in combination with neural stem/progenitor cell transplantation and rehabilitation. This comprehensive therapeutic strategy achieved significant improvements in host-derived neuronal and oligodendrocyte differentiation at the SCI epicenter and promoted axonal regeneration even in the chronically injured spinal cord. The elongated axons established functional electrical connections, contributing to significant enhancements in locomotor mobility when compared with animals treated with transplantation and rehabilitation. As a result, our combined transplantation, Sema3Ai, and rehabilitation treatment have the potential to serve as a critical step forward for chronic SCI patients, improving their ability to regain motor function.

The TGFβ1/SMADs/Snail1 signaling axis mediates pericyte-derived fibrous scar formation after spinal cord injury

AimsThe deposition of fibrous scars after spinal cord injury (SCI) affects axon regeneration and the recovery of sensorimotor function. It has been reported that microvascular pericytes in the neurovascular unit are the main source of myofibroblasts after SCI, but the specific molecular targets that regulate pericyte participation in the formation of fibrous scars remain to be clarified. MethodsIn this study, a rat model of spinal cord dorsal hemisection injury was used. After SCI, epigallocatechin gallate (EGCG) was intraperitoneally injected to block the TGFβ1 signaling pathway or LV-Snail1-shRNA was immediately injected near the core of the injury using a microsyringe to silence Snail1 expression. Western blotting and RT-qPCR were used to analyze protein expression and transcription levels in tissues. Nissl staining and immunofluorescence analysis were used to analyze neuronal cell viability, scar tissue, and axon regeneration after SCI. Finally, the recovery of hind limb function after SCI was evaluated. ResultsThe results showed that targeted inhibition of Snail1 could block TGFβ1-induced pericyte-myofibroblast differentiation in vitro. In vivo experiments showed that timely blockade of Snail1 could reduce fibrous scar deposition after SCI, promote axon regeneration, improve neuronal survival, and facilitate the recovery of lower limb motor function. ConclusionIn summary, Snail1 promotes the deposition of fibrous scars and inhibits axonal regeneration after SCI by inducing the differentiation of pericytes into myofibroblasts. Snail1 may be a promising therapeutic target for SCI.

The Effect of Therapeutic Exercise on Body Weight Distribution, Balance, and Stifle Function in Dogs following Stifle Injury.

Stifle injury is common in the companion dog population, affecting weight bearing, neuromuscular control, and balance. Therapeutic exercises after stifle injury seem to be effective, but high-quality research evaluating the effects is lacking. This randomized controlled trial evaluated the effects of a 12-week progressive therapeutic home exercise protocol on three-legged standing, targeting balance and postural- and neuromuscular control and disability in dogs with stifle injury. Thirty-three dogs with stifle injury were randomly allocated to intervention (n = 18) and control groups (n = 15), both receiving a standard rehabilitation protocol. Additionally, the intervention group received a progressive therapeutic exercise protocol. The outcome measures were static body weight distribution between hindlimbs, balance control, the canine brief pain inventory, and the Finnish canine stifle index. Both groups improved after the intervention period, but the group using the progressive therapeutic exercise protocol improved to a greater extent regarding static body weight distribution between the hindlimbs (I: median = 2.5%, IQR = 1.0-4.5; C: median = 5.5%, IQR = 3.0-8.8), pain-related functional disability (I: median = 0.0, IQR = 0.0-0.2; C: median = 0.9, IQR = 0.1-1.8), and stifle function (I: median = 25.0, IQR = 9.4-40.6; C: median = 75.0, IQR = 31.3-87.5), with intermediate to strong effects. These clinically relevant results indicate that this home exercise program can improve hindlimb function and restore neuromuscular control.

An Open-Source 3D-Printed Hindlimb Stabilization Apparatus for Reliable Measurement of Stimulation-Evoked Ankle Flexion in Rat.

Currently there are numerous methods to evaluate peripheral nerve stimulation interfaces in rats, with stimulation-evoked ankle torque being one of the most prominent. Commercial rat ankle torque measurement systems and custom one-off solutions have been published in the literature. However, commercial systems are proprietary and costly and do not allow for customization. One-off lab-built systems have required specialized machining expertise, and building plans have previously not been made easily accessible. Here, detailed building plans are provided for a low-cost, open-source, and basic ankle torque measurement system from which additional customization can be made. A hindlimb stabilization apparatus was developed to secure and stabilize a rat's hindlimb, while allowing for simultaneous ankle-isometric torque and lower limb muscle electromyography (EMG). The design was composed mainly of adjustable 3D-printed components to accommodate anatomical differences between rat hindlimbs. Additionally, construction and calibration procedures of the rat hindlimb stabilization apparatus were demonstrated in this study. In vivo torque measurements were reliably acquired and corresponded to increasing stimulation amplitudes. Furthermore, implanted leads used for intramuscular EMG recordings complemented torque measurements and were used as an additional functional measurement in evaluating the performance of a peripheral nerve stimulation interface.In conclusion, an open-source and noninvasive platform, made primarily with 3D-printed components, was constructed for reliable data acquisition of evoked motor activity in rat models. The purpose of this apparatus is to provide researchers a versatile system with adjustable components that can be tailored to meet user-defined experimental requirements when evaluating motor function of the rat hindlimbs.

Decellularized extracellular matrix scaffold seeded with adipose-derived stem cells promotes neurorestoration and functional recovery after spinal cord injury through Wnt/β-catenin signaling pathway regulation

Spinal cord injury (SCI) causes tissue destruction and neuronal apoptosis, which impede neural function recovery. Therefore, promoting neuronal regeneration and neural pathway reconstruction is crucial. In this study, a novel and facile decellularized extracellular matrix (dECM) scaffold seeded with adipose-derived stem cells (ADSCs) (dECM scaffolds/ADSCs) was reported. The dECM scaffold maintained the original three-dimensional network structure of spinal cord tissue and contained various small pores. In vitro studies demonstrated that dECM scaffolds exhibited excellent biocompatibility, facilitated efficient adhesion and proliferation of ADSCs, and promoted the secretion of neurotrophin-3 and neuronal differentiation in the microenvironment after SCI. In vivo studies further showed that dECM scaffolds/ADSCs could alleviate inflammatory and apoptotic reactions, providing a favorable microenvironment for promoting endogenous nerve regeneration rather than glial scars formation, ultimately achieving recovery of hind limb function in rats. Notably, ICG-001 effectively reversed the therapeutic effect of dECM scaffolds/ADSCs, proving that dECM scaffolds/ADSCs promoted functional recovery after SCI by regulating the Wnt/β-catenin signaling pathway. Overall, dECM scaffolds/ADSCs can simulate the physiological characteristics of the spinal cord and exert neurorestorative potential, providing a new therapeutic strategy for SCI.