Changes in 5-HT1F receptor expression in rats with spasticity following spinal cord injury.

Spasticity is a common complication in patients with spinal cord injury (SCI) and adversely affects patients' quality of life. Little is known about the distribution of the serotonin 1F receptor (5-HT1FR) in the spinal cord, especially in relation to the spasticity caused by SCI. Adult male Wistar rats were divided into a sham-operation group and spinalized group. SCI-induced spasticity was caused by spinal transection at the second sacral segment. The spinal cord below the transection was obtained at the end of the experiment. The expression and distribution of 5-HT1FR in the spinal cord were analyzed. The results showed that the expression of 5-HT1FR (mRNA and protein) exhibited the same downward trend after spinal transection and reached the lowest expression level at 2 and 5 days, respectively. The expression of 5-HT1FR (mRNA and protein) thereafter gradually approached the levels in the sham-operation group after 60 days. Immunostaining suggested that 5-HT1FR showed particularly strong expression in the ventral horn (VH) region. The time course of 5-HT1FR mRNA downregulation is positively correlated with the development of tail spasticity after sacral spinal cord transection. There may be a connection between 5-HT1FR and the occurrence of spasticity, but elucidation of the specific mechanism needs further experimental verification.

Proteomics Reveals the Effect of Low-Intensity Focused Ultrasound on Spasticity After Spinal Cord Injury.

AIM: To explore the efficacy and possible mechanisms of Low-intensity focused ultrasound (LIFU) in alleviating spasticity caused by Spinal cord injury (SCI). MATERIAL AND METHODS: We selected male Sprague?Dawley rats as subjects and performed transverse injuries on the T9 vertebra of their spinal cord (SC) to build SCI. On the 7th day after SCI, LIFU treatment was performed below the SCI segment once a day for 20 min, for 4 consecutive weeks. During treatment, a pressure sensor was used to assess the degree of spasticity. After treatment, the SC tissues from the treatment sites of the SCI+LIFU(-) and SCI+LIFU(+) groups were extracted, and high-throughput sequencing was performed to identify the changes in proteomics. In addition, expression of the growth associated protein 43 (Gap43) was validated by western blotting. RESULTS: The behavioral results suggested that after 2 weeks of SCI, the rats were significantly induced to have a spastic reaction (p < 0.05), while after 4 weeks of LIFU treatment, the spastic response of rats was significantly improved (p < 0.05). Western blot analysis showed a significant increase in Gap43 expression in the SCI+LIFU(-) group compared with the sham group, whereas after 4 weeks of LIFU treatment, Gap43 protein expression was significantly decreased (p < 0.05). CONCLUSION: The results of this study showed that LIFU is an alternative treatment that can effectively relieve spastic reactions caused by SCI, possibly by reducing abnormal neuroplasticity or axon regeneration below the SCI segment.

The J Domain of Sacsin Disrupts Intermediate Filament Assembly.

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS) is caused by mutation in the SACS gene resulting in loss of function of the protein sacsin. A key feature is the formation of abnormal bundles of neurofilaments (NF) in neurons and vimentin intermediate filaments (IF) in cultured fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons. Having studied the mechanism using NF assembled in vitro from purified NF proteins, we report that the SacsJ domain interacts with NF proteins to disassemble NFL filaments, and to inhibit their initial assembly. A cell-penetrating peptide derived from this domain, SacsJ-myc-TAT was efficient in disassembling NF bundles in cultured Sacs-/- motor neurons, restoring the NF network; however, there was some loss of vimentin IF and NF in cultured Sacs+/+ fibroblasts and motor neurons, respectively. These results suggest that sacsin through its SacsJ domain is a key regulator of NF and vimentin IF networks in cells.

Molecular Identification of Pro-Excitogenic Receptor and Channel Phenotypes of the Deafferented Lumbar Motoneurons in the Early Phase after SCT in Rats.

Spasticity impacts the quality of life of patients suffering spinal cord injury and impedes the recovery of locomotion. At the cellular level, spasticity is considered to be primarily caused by the hyperexcitability of spinal α-motoneurons (MNs) within the spinal stretch reflex circuit. Here, we hypothesized that after a complete spinal cord transection in rats, fast adaptive molecular responses of lumbar MNs develop in return for the loss of inputs. We assumed that early loss of glutamatergic afferents changes the expression of glutamatergic AMPA and NMDA receptor subunits, which may be the forerunners of the developing spasticity of hindlimb muscles. To better understand its molecular underpinnings, concomitant expression of GABA and Glycinergic receptors and serotoninergic and noradrenergic receptors, which regulate the persistent inward currents crucial for sustained discharges in MNs, were examined together with voltage-gated ion channels and cation-chloride cotransporters. Using quantitative real-time PCR, we showed in the tracer-identified MNs innervating extensor and flexor muscles of the ankle joint multiple increases in transcripts coding for AMPAR and 5-HTR subunits, along with a profound decrease in GABAAR, GlyR subunits, and KCC2. Our study demonstrated that both MNs groups similarly adapt to a more excitable state, which may increase the occurrence of extensor and flexor muscle spasms.

The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes.

Acupuncture improves the structure of spastic muscle and decreases spasticity by enhancing GABA, KCC2, and GABAAγ2 in the brainstem in rats after ischemic stroke.

BACKGROUND: Increasing data show that structural changes of spastic muscle and hyperexcitability of reticulospinal tract (RST) are involved in the pathogenesis of spasticity after stroke (SAS). Our previous study has indicated that the anti-spastic effect of acupuncture, especially waggle needling (WN, a multiple directional needling method with joint movement), on SAS rats was related to the KCC2-GABAA pathway in cerebral cortex. Furthermore, as a peripheral stimulation to treat upper motor neuron injury-related spasticity, acupuncture's effect on peripheral spastic muscles and inhibitory neurotransmitters in the brainstem, the origin of the RST, should be further clarified. This study aimed to examine the effect of acupuncture on the structure of spastic muscle and on the KCC2-GABAA pathway in the brainstem of SAS rats. METHODS: Middle cerebral artery occlusion (MCAO) or a sham operation were conducted in SD rats to establish SAS and control models. Behavioral assays, muscle myosin ATPase staining, and molecular biology technologies were used to compare different groups. RESULTS: In SAS models, hindlimb motor ability was decreased, neurologic deficits and spasticity were induced, the proportion of type I muscle fibers in spastic muscle was increased, and the expressions of γ-aminobutyric acid (GABA), KCC2, and the GABAAγ2 subunit of the pentameric GABAA receptor in the brainstem were decreased. Acupuncture including WN and perpendicular needling (PN) reversed these effects of MCAO. Furthermore, the therapeutic effect of WN was better than that of PN. CONCLUSIONS: Acupuncture after MCAO improves the structure of spastic muscle and decreases spasticity probably at least partly by enhancing GABA, KCC2, and GABAAγ2 in the brainstem in SAS rats.

Body Weight-Supported Treadmill Training Ameliorates Motoneuronal Hyperexcitability by Increasing GAD-65/67 and KCC2 Expression via TrkB Signaling in Rats with Incomplete Spinal Cord Injury.

Spasticity is a typical consequence after spinal cord injury (SCI). The critical reasons are reducing the synthesis of Gamma-Aminobutyric Acid (GABA), glycine and potassium chloride co-transporter 2 (KCC2) inside the distal spinal cord. The current work aimed to test whether exercise training could increase the expression of glutamic acid decarboxylase 65/67 (GAD-65/67, the key enzymes in GABA synthesis) and KCC2 in the distal spinal cord via tropomyosin-related kinase B (TrkB) signaling. The experimental rats were randomly assigned to the following five groups: Sham, SCI/phosphate-buffered saline (PBS), SCI-treadmill training (TT)/PBS, SCI/TrkB-IgG, and SCI-TT/TrkB-IgG. After that, the model of T10 contusion SCI was used, then TrkB-IgG was used to prevent TrkB activity at 7 days post-SCI. Body weight-supported treadmill training started on the 8th day post-SCI for four weeks. The Hmax/Mmax ratio and the rate-dependent depression of H-reflex were used to assess the excitability of spinal motoneuronal networks. Western blotting and Immunohistochemistry techniques were utilized for measuring the expression of GAD-65, GAD-67, and KCC2. The findings revealed that exercise training could reduce motoneuronal excitability and boost GAD-65, GAD-67, and KCC2 production in the distal region of the spinal cord after SCI. The effects of exercise training were decreased after the TrkB signaling was inhibited. The present exploration demonstrated that exercise training increases GAD-65, GAD-67, and KCC2 expression in the spinal cord via TrkB signaling and that this method could also improve rats with motoneuronal hyperexcitability and spasticity induced by incomplete SCI.

Acupuncture alleviates spinal hyperreflexia and motor dysfunction in post-ischemic stroke rats with spastic hypertonia via KCC2-mediated spinal GABAA activation.

The majority of patients simultaneously develop motor dysfunction and spastic hypertonia after ischemic strokes, which can be associated with an increasing trend in motor impairments, seriously impeding the rehabilitation process. Evidence suggests that some deficits in the KCC2 expression in the spinal cord along with maladaptive endogenous plasticity via GABAA receptors are often involved in the pathology of spastic hypertonia after a stroke. In this respect, acupuncture has been commonly used in clinical settings for post-stroke patients' rehabilitation. Nevertheless, the mechanism of the modulating activity of this alternative medicine in the spinal pathways to relieve spasticity and improve functional recovery after a stroke has still remained unclear. Utilizing laser speckle imaging, functional assessments (viz. neurologic function scale, muscular tension scale, foot balance test, and gait analysis), H-reflex recording, TTC, Western blotting, RT-qPCR, ELISA, and immunofluorescence molecular assay, the study results illustrated that acupuncture could significantly alleviate the spinal hyperreflexia, decrease muscle tone, and enhance locomotor function by elevating the GABA, KCC2, and GABAAγ2 expressions in the lumbar spine of a rat model of post-ischemic stroke with spastic hypertonia. Furthermore, the KCC2 antagonist DIOA abolished the benefits induced by this practice. Overall, the data revealed that acupuncture is a promising therapeutic approach for spastic hypertonia after a stroke, and the positive outcomes in this sense could be achieved via activating the KCC2-mediated spinal GABAA signaling pathway.

Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

Hsp90 Inhibition: A Promising Therapeutic Approach for ARSACS.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease caused by mutations in the SACS gene, encoding the 520 kDa modular protein sacsin, which comprises multiple functional sequence domains that suggest a role either as a scaffold in protein folding or in proteostasis. Cells from patients with ARSACS display a distinct phenotype including altered organisation of the intermediate filament cytoskeleton and a hyperfused mitochondrial network where mitochondrial respiration is compromised. Here, we used vimentin bundling as a biomarker of sacsin function to test the therapeutic potential of Hsp90 inhibition with the C-terminal-domain-targeted compound KU-32, which has demonstrated mitochondrial activity. This study shows that ARSACS patient cells have significantly increased vimentin bundling compared to control, and this was also present in ARSACS carriers despite them being asymptomatic. We found that KU-32 treatment significantly reduced vimentin bundling in carrier and patient cells. We also found that cells from patients with ARSACS were unable to maintain mitochondrial membrane potential upon challenge with mitotoxins, and that the electron transport chain function was restored upon KU-32 treatment. Our preliminary findings presented here suggest that targeting the heat-shock response by Hsp90 inhibition alleviates vimentin bundling and may represent a promising area for the development of therapeutics for ARSACS.

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs-/- larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

Conditional RAC1 knockout in motor neurons restores H-reflex rate-dependent depression after spinal cord injury.

A major complication with spinal cord injury (SCI) is the development of spasticity, a clinical symptom of hyperexcitability within the spinal H-reflex pathway. We have previously demonstrated a common structural motif of dendritic spine dysgenesis associated with hyperexcitability disorders after injury or disease insults to the CNS. Here, we used an adeno-associated viral (AAV)-mediated Cre-Lox system to knockout Rac1 protein expression in motor neurons after SCI. Three weeks after AAV9-Cre delivery into the soleus/gastrocnemius of Rac1-"floxed" adult mice to retrogradely infect spinal alpha-motor neurons, we observed significant restoration of RDD and reduced H-reflex excitability in SCI animals. Additionally, viral-mediated Rac1 knockdown reduced presence of dendritic spine dysgenesis on motor neurons. In control SCI animals without Rac1 knockout, we continued to observe abnormal dendritic spine morphology associated with hyperexcitability disorder, including an increase in mature, mushroom dendritic spines, and an increase in overall spine length and spine head size. Taken together, our results demonstrate that viral-mediated disruption of Rac1 expression in ventral horn motor neurons can mitigate dendritic spine morphological correlates of neuronal hyperexcitability, and reverse hyperreflexia associated with spasticity after SCI. Finally, our findings provide evidence of a putative mechanistic relationship between motor neuron dendritic spine dysgenesis and SCI-induced spasticity.

Enhancing KCC2 activity decreases hyperreflexia and spasticity after chronic spinal cord injury.

After spinal cord injury (SCI), the majority of individuals develop spasticity, a debilitating condition involving involuntary movements, co-contraction of antagonistic muscles, and hyperreflexia. By acting on GABAergic and Ca2+-dependent signaling, current anti-spastic medications lead to serious side effects, including a drastic decrease in motoneuronal excitability which impairs motor function and rehabilitation efforts. Exercise, in contrast, decreases spastic symptoms without decreasing motoneuron excitability. These functional improvements coincide with an increase in expression of the chloride co-transporter KCC2 in lumbar motoneurons. Thus, we hypothesized that spastic symptoms can be alleviated directly through restoration of chloride homeostasis and endogenous inhibition by increasing KCC2 activity. Here, we used the recently developed KCC2 enhancer, CLP257, to evaluate the effects of acutely increasing KCC2 extrusion capability on spastic symptoms after chronic SCI. Sprague Dawley rats received a spinal cord transection at T12 and were either bike-trained or remained sedentary for 5 weeks. Increasing KCC2 activity in the lumbar enlargement improved the rate-dependent depression of the H-reflex and reduced both phasic and tonic EMG responses to muscle stretch in sedentary animals after chronic SCI. Furthermore, the improvements due to this pharmacological treatment mirror those of exercise. Together, our results suggest that pharmacologically increasing KCC2 activity is a promising approach to decrease spastic symptoms in individuals with SCI. By acting to directly restore endogenous inhibition, this strategy has potential to avoid severe side effects and improve the quality of life of affected individuals.

Generation and Characterization of a New Resistance to Thyroid Hormone Mouse Model with Thyroid Hormone Receptor Alpha Gene Mutation.

Background: In humans, resistance to thyroid hormone (RTH) caused by mutations in the thyroid hormone receptor alpha (THRA) gene, RTHα, manifests as tissue-specific hypothyroidism and circulating thyroid hormone levels exhibit hypothyroid-like clinical features. Before the identification of patients with RTHα, several Thrα1 knock-in mouse models were generated to clarify the function of TRα1. However, the phenotypes of these mice were not consistent with the clinical presentation of RTHα in humans. For the present study, we generated an RTHα mouse model that carries the Thra1E403X mutation found in human RTHα patients. Here, we report the gross phenotypes of this mouse RTHα model. Methods: Traditional homologous recombination gene targeting techniques were used to introduce a mutation (Thra1E403X) in the mouse Thra gene. The phenotypes of the resulting mice were studied and compared with clinical features observed for RTHα with THRAE403X. Results: Thrα1E403X/E403X homozygous mice exhibited severe neurological phenotypes, such as spasticity and motor ataxia, which were similar to those observed in endemic cretinism. Thrα1E403X/+ heterozygous mice reproduced most clinical manifestations of patient with RTHα, such as a normal survival rate and male fertility, as well as delayed postnatal growth and development, neurological and motor coordination deficits, and anemia. The mice had typical thyroid function with a modest increase in serum triiodothyronine (T3) levels, a low thyroxine (T4)/T3 ratio, and low reverse T3 (rT3) levels. Conclusions: The Thrα1E403X/+ mice faithfully recapitulate the clinical features of human RTHα and thus can provide a useful tool to dissect the role of TRα1 in development and to determine the pathological mechanisms of RTHα.

Altered monoaminergic levels, spasticity, and balance disability following repetitive blast-induced traumatic brain injury in rats.

Spasticity and balance disability are major complications following traumatic brain injury (TBI). Although monoaminergic inputs provide critical adaptive neuromodulations to the motor system, data are not available regarding the levels of monoamines in the brain regions related to motor functions following repetitive blast TBI (bTBI). The objective of this study was to determine if mild, repetitive bTBI results in spasticity/balance deficits and if these are correlated with altered levels of norepinephrine, dopamine, and serotonin in the brain regions related to the motor system. Repetitive bTBI was induced by a blast overpressure wave in male rats on days 1, 4, and 7. Following bTBI, physiological/behavioral tests were conducted and tissues in the central motor system (i.e., motor cortex, locus coeruleus, vestibular nuclei, and lumbar spinal cord) were collected for electrochemical detection of norepinephrine, dopamine, and serotonin by high-performance liquid chromatography. The results showed that norepinephrine was significantly increased in the locus coeruleus and decreased in the vestibular nuclei, while dopamine was significantly decreased in the vestibular nuclei. On the other hand, serotonin was significantly increased in the motor cortex and the lumbar spinal cord. Because these monoamines play important roles in regulating the excitability of neurons, these results suggest that mild, repetitive bTBI-induced dysregulation of monoaminergic inputs in the central motor system could contribute to spasticity and balance disability. This is the first study to report altered levels of multiple monoamines in the central motor system following acute mild, repetitive bTBI.

1-O-Alkylglycerol accumulation reveals abnormal ether glycerolipid metabolism in Sjögren-Larsson syndrome.

Sjögren-Larsson syndrome (SLS) is an inherited metabolic disease characterized by ichthyosis, spasticity, intellectual disability and deficient oxidation and accumulation of of fatty aldehydes and alcohols. We investigated whether excess fatty alcohols in SLS are diverted into biosynthesis of ether glycerolipids (eGLs) by measuring the 1-O-alkylglycerol (AG) backbone of eGLs in stratum corneum, plasma and red blood cells (RBCs). In all tissues, saturated and monounsaturated AGs were detected. In stratum corneum from SLS patients, saturated AGs (C15-C20) were increased 97-fold (range: 86- to 169-fold) compared to controls. AGs were largely (67 ± 9%) derived from neutral esterified eGLs (i.e. alkyl-diacylglyerol) and free non-esterified AGs (28 ± 10%), but very little from plasmalogens (3 ± 5%). Plasma from SLS patients had 2-fold more C18:0-AG (p < 0.005) and 40% less C16:1-AG (p < 0.01) than controls but the total concentration of AGs was not increased, and the AG profile in RBCs from SLS subjects was normal. All AGs were profoundly reduced in plasma and RBCs from patients with Zellweger spectrum disorder, who have impaired eGL (i.e. plasmalogen) synthesis. The striking accumulation of AGs in stratum corneum of SLS patients constitutes a novel lipid biomarker for this disease, and may contribute to the pathogenesis of the ichthyosis. Measurement of AGs is a simple and convenient method to assess global synthesis of eGLs and potentially identify patients with defects in their metabolism.

Botulinum Toxin Type A Possibly Affects Cav3.2 Calcium Channel Subunit in Rats with Spinal Cord Injury-Induced Muscle Spasticity.

INTRODUCTION: Spinal cord injury (SCI) often causes muscle spasticity, which can be inhibited by using calcium channel blocker. Botulinum toxin type A (BoT-A) shows therapeutic efficacy on spasticity and may exert inhibitory effects on the calcium channel. METHODS: A rat model with muscle spasticity was established after SCI via contusion and compression. Different concentrations (0, 1, 3 and 6 U/kg) of BoT-A Botox were injected in the extensor digitorum longus (EDL) muscles of the right hindlimb in the muscle spasticity model. The changes of muscle spasticity and calcium level in EDL muscles were measured after the establishment of SCI-induced spasticity. Cav3.2 calcium channel subunit and its mutant (M1560V) were analyzed using Western blot before (input) or after immunoprecipitation with anti-FLAG antibody, and their currents were measured in motoneurons by using whole-cell voltage clamp recordings. RESULTS: SCI induced muscle spasticity, whereas calcium level in EDL muscles and expression of Cav3.2 was increased in the SCI model when compared with the sham group (p < 0.05). BoT-A Botox treatment significantly reduced muscle spasticity and calcium level in EDL muscles and Cav3.2 expression in a dose-dependent way (p < 0.05). The ratio of biotinylated to total Cav3.2 was reduced in the mutant (M1560V) of Cav3.2 and lower than that in the wild Cav3.2. BoT-A Botox intervention also reduced the current values of calcium channel and the ratio in a dose-dependent way (p < 0.05). DISCUSSION: BoT-A Botox possibly attenuates SCI-induced muscle spasticity by affecting the expression of Cav3.2 calcium channel subunit in the rat models. There may be multiple mechanisms for the function of BoT-A Botox. Further work is needed to be done to address these issues.

Waggle needling wields preferable neuroprotective and anti-spastic effects on post-stroke spasticity rats by attenuating γ-aminobutyric acid transaminase and enhancing γ-aminobutyric acid.

Waggle needling, a classical anti-spastic needling technique characterized by combination of acupuncture with joint movement, has gained increasing popularity of spasticity treatment in China. This study was designed to compare the anti-spastic effect of waggle needling to the routine needling and to explore its underlying mechanism. We established post-stroke spasticity model based on ischemia stroke operation (middle cerebral artery occlusion). Rats were divided into six groups: normal control group, sham-operated control group, ischemia stroke model group, waggle needling group, routine needling group and baclofen group. Neurological function and muscle tone were assessed by the Zea Longa score and modified Ashworth scale, respectively. Indirect muscle tone was testified with electrophysiological recording. Cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride staining. The concentrations and expressions of γ-aminobutyric acid transaminase (GABAT) and γ-aminobutyric acid (GABA) were detected by enzyme-linked immunosorbent assay and western blot assay. Waggle needling markedly alleviated neurological deficits, decreased cerebral infarction and eased muscle tone; simultaneously, attenuated GABAT and enhanced GABA expression in the cortical infarct regions in comparison with the routine needling (P < 0.01), yet showed similar therapeutic effect to the baclofen group (P > 0.05). These results preliminary supported that waggle needling as a potential promising non-pharmacological intervention for the treatment of cerebral ischemia and spasticity.

Alteration of glycinergic receptor expression in lumbar spinal motoneurons is involved in the mechanisms underlying spasticity after spinal cord injury.

Spasticity is a disabling motor disorder affecting 70% of people with brain and spinal cord injury. The rate-dependent depression (RDD) of the H reflex is the only electrophysiological measurement correlated with the degree of spasticity assessed clinically in spastic patients. Several lines of evidence suggest that the mechanism underlying the H reflex RDD depends on the strength of synaptic inhibition through GABAA (GABAAR) and glycine receptors (GlyR). In adult rats with spinal cord transection (SCT), we studied the time course of the expression of GABAAR and GlyR at the membrane of retrogradely identified Gastrocnemius and Tibialis anterior motoneurons (MNs) 3, 8 and 16 weeks after injury, and measured the RDD of the H reflex at similar post lesion times. Three weeks after SCT, a significant decrease in the expression of GABAA and GlyR was observed compared to intact rats, and the H-reflex RDD was much less pronounced than in controls. Eight weeks after SCT, GlyR values returned to normal. Simultaneously, we observed a tendency to recover normal RDD of the H reflex at higher frequencies. We tested whether an anti-inflammatory treatment using methylprednisolone performed immediately after SCT could prevent alterations in GABAA/glycine receptors and/or the development of spasticity observed 3 weeks after injury. This treatment restored control levels of GlyR but not the expression of GABAAR, and it completely prevented the attenuation of RDD. These data strongly suggest that alteration of glycinergic inhibition of lumbar MNs is involved in the mechanisms underlying spasticity after SCI.