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.

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.

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.

Venous thromboembolism due to hyperhomocysteinaemia and tuberculosis.

An 18-year-old male presented to our hospital with complaints of episodic abdominal pain, dry cough and right pleuritic chest pain. He was diagnosed as a case of right tuberculous pleural effusion on the basis of the pleural fluid Genexpert report of Mycobacterium tuberculosis detected sensitive to rifampicin and was started on antituberculous therapy. Forty-five days later, he presented with acute onset breathlessness, swelling of the right leg, streaky haemoptysis and a fresh left-sided pleural effusion. Evaluation revealed venous thromboembolism (right lower lobar segment pulmonary embolism with right leg deep vein thrombosis). Workup for malignancy was negative. However, he had vitamin B12 deficiency with increased homocysteine levels and heterozygous mutation of the MTHFR gene at A1298C. He was treated with optimal anticoagulation, vitamin B12 supplementation and antitubercular treatment. This is a rare combination of events perhaps related to the MTHFR gene mutation.

[Effects of Shaoyao Gancao decoction on contents of amino acids and expressions of receptors in brains of spastic paralysis rats].

To explore the effects of Shaoyao Gancao decoction on contents of amino acids and expressions of receptors in the brains of spastic paralysis rats, the spastic paralysis rat models of stroke convalescence were made by line tethering method. Baclofen was used as the control group, and the experiment group received Shaoyao Gancao decoction at 3∶1 proportions. After 3 weeks, the neurobehavioral scores, muscular tension and pain threshold were measured and compared. High performance liquid chromatography (HPLC) was used to detect the contents of GABA, Gly, Glu, Asp in cerebral cortex. The protein expressions of GABA receptors Aα1, B； NMDA receptor NR1, NR2A and NR2B in cerebral cortex were determined by immunohistochemistry assay. The results showed that the Shaoyao Gancao decoction at 3∶1 proportion could improve the spastic paralysis state after stroke, significantly improve neurological symptoms (P<0.01), decrease muscular tension (P<0.01) and improve pain threshold (P<0.05) as compared with model group. Simultaneously, the contents of inhibitory amino acids GABA and Gly were increased significantly (P<0.01), while with a decrease tendency in excitatory amino acids Glu and Asp (with no statistical significance). In addition, it could significantly increase the protein expressions of neurotransmitter GABA receptors Aα1, and B (P<0.05); reduce the expressions of neurotransmitter NMDA receptors NR1, NR2A and NR2B (P<0.05). These results suggested that the Shaoyao Gancao decoction at 3：1 proportion could effectively relieve spasm and pain. The mechanism might be associated with increasing the contents of inhibitory amino acids and increasing the expressions of their receptors in spastic paralysis rats after stroke, which would consequently enhance the signal transduction of inhibitory amino acids. Meanwhile, there was a decrease tendency in excitatory amino acids, although no significant effect was observed, and it could suppress the expressions of excitatory amino acids receptors, thus weaken the excitatory signal transduction. Thereby the neurotoxicity was relieved eventually. These findings indicated that Shaoyao Gancao decoction could regulate the balance of neurotransmitter system to relieve the spasticity, and eventually achieve tendon tonifying and spasm relieving effect.

High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice.

BACKGROUND: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions. OBJECTIVE: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism. DESIGN: Folic acid-supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr(+/+) and Mthfr(+/-) mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined. RESULTS: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr(+/-) mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr(+/-) livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr(+/-) mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile. CONCLUSIONS: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.

Effect of catgut implantation at acupoints on GABA(B) and mGluR1 expressions in brain stem of rats with spasticity after stroke. .

OBJECTIVE: To investigate the effect of catgut implantation at acupoints on the expressions of γ-amino butyric acid B receptor (GABA(B)) and metabotropic glutamate receptor 1 (mGluR1) in the brain stem of rats with spasticity after stroke. METHODS: In total, 60 male Sprague-Dawley rats were randomly divided into three groups: a sham group (n = 10), a model group (n = 25) and a treatment group (n = 25). The rats in both the model group and the treatment group were subjected to middle cerebral artery occlusion to establish a model of focal cerebral ischemia. Rats with limb-spasm met the inclusion criteria. Only the left carotid artery was isolated in sham group rats. Three days after modeling, the treatment group was subjected to catgut implantation at Dazhui (GV 14), Guanyuan (CV 4), and Zhongwan (CV 12). Neurological deficit symptoms were assessed with the Zea-Longa neurological deficit score. The Modified Ashworth Scale (MAS), and isolated muscle tone were used to evaluate spasticity before and after treatment. Immunohistochemistry was applied to determine the expression of GABA(B) and mGluR1 in the rat brain stem after treatment. RESULTS: After treatment, neural impairment symptoms had significantly improved in the treatment group when compared to the model group (P < 0.05). Both MAS and isolated muscle tone in the treatment group were significantly decreased when compared with the model group (P < 0.05), and were also lower than before treatment. GABA(B) expression was significantly higher and mGluR1 was lower in the treatment group when compared with the model group (P < 0.01 and P < 0.05, respectively). CONCLUSION: Catgut implantation at Dazhui (GV14), Guanyuan (CV 4), and Zhongwan (CV 12), can relieve limb spasticity by increasing the expression of GABA(B) and reducing the expression of mGluR1 in the brain stem of rats after stroke.

Endocannabinoid pathways and their role in multiple sclerosis-related muscular dysfunction.

Endocannabinoids are endogenous agonists of the mammalian cannabinoid receptors CB(1) and CB(2), and they appear to be produced in tissues as an adaptive reaction to re-establish normal homeostasis when this is acutely altered. However, the production of endocannabinoids can be altered pathologically. The two most widely studied endocannabinoids are anandamide and 2-arachidonoyl glycerol. The levels of these endogenous modulators are regulated in different and sometimes opposing ways, and alterations in cerebrospinal fluid and/or spinal cord levels have been documented in animal models of neurodegenerative diseases and in samples from patients with multiple sclerosis (MS). Modulation of the endocannabinoid system has been shown to have therapeutic potential in a number of disease states. Sativex(®) (nabiximols, USAN name) contains the two main phytocannabinoids from Cannabis sativa, tetrahydrocannabinol and cannabidiol in a 1:1 ratio, and it acts as an endocannabinoid system modulator. In an experimental mouse model of MS-related spasticity, Sativex dose-dependently improved hind limb flexion/stiffness and a dosage of 10 mg/kg was shown to be as effective as the most widely established anti-spasticity treatment baclofen (5 mg/kg). These findings with Sativex are very promising and offer encouragement for MS patients, the majority of whom will develop spasticity-related disabling and recalcitrant symptoms. Furthermore, research into the endocannabinoid system may offer potential in other neurodegenerative, inflammatory and pain disorders.

Sativex(®) (tetrahydrocannabinol + cannabidiol), an endocannabinoid system modulator: basic features and main clinical data.

Sativex(®) (nabiximols, USAN name) oromucosal spray contains the two main active constituents of Cannabis sativa, tetrahydrocannabinol and cannabidiol in a 1:1 molecular ratio, and acts as an endocannabinoid system modulator. Randomized, controlled clinical trials of Sativex as add-on therapy provide conclusive evidence of its efficacy in the treatment of more than 1500 patients with multiple sclerosis (MS)-related resistant spasticity. The primary end point in clinical trials was the mean change from baseline in the 0-10 numerical rating scale (NRS) spasticity score. The first pivotal clinical trial included 189 patients treated for 6 weeks with Sativex (n = 124) or placebo (n = 65). At study end, there was a significant reduction from baseline in patient-recorded NRS spasticity scores with Sativex compared with placebo (-1.18 vs -0.63; p = 0.048). In the second pivotal trial, 337 patients with MS-related resistant spasticity received Sativex (n = 167) or placebo (n = 170) over a 15-week period. In the per-protocol analysis (79% of the patient population), mean baseline NRS spasticity score was reduced significantly in patients receiving Sativex compared with placebo: -1.3 versus -0.8 points (p = 0.035). The third pivotal clinical trial, evaluating the sustained efficacy of Sativex, had a two-phase study design: in phase A (n = 572), 47% of patients were initial responders (improvement ≥ 20%) after 4 weeks of single-blind Sativex treatment who then entered phase B, a randomized, double-blind, 12-week placebo comparison. At the end of phase B, the change in NRS spasticity score improved by a further 0.04 units in initial responders treated with Sativex, but decreased by 0.81 units in placebo recipients (p = 0.0002). Significant improvements in quality-of-life measures from baseline to week 16 were also observed in patients receiving Sativex. The most common treatment-related adverse events with Sativex were mild-to moderate and transient episodes of dizziness, fatigue or somnolence. Sativex does not exhibit the side effects typically associated with recreational cannabis use and there are no signs of drug tolerance or withdrawal syndrome, or any evidence of drug misuse or abuse. Sativex oromucosal spray appears to be a useful and welcomed option for the management of resistant spasticity in MS patients. Although the management of MS has been improved by the availability of disease-modifying agents that target the underlying pathophysiological processes of the disease, a clear need remains for more effective symptomatic treatments, especially as regards MS-related spasticity and pain.

Suppressing the excitability of spinal motoneurons by extracellularly applied electrical fields: insights from computer simulations.

The effect of extracellularly applied electrical fields on neuronal excitability and firing behavior is attributed to the interaction between neuronal morphology and the spatial distribution and level of differential polarization induced by the applied field in different elements of the neuron. The presence of voltage-gated ion channels that mediate persistent inward currents (PICs) on the dendrites of spinal motoneurons enhances the influence of electrical fields on the motoneuronal firing behavior. The goal of the present study was to investigate, with a realistic motoneuron computer model, the effects of extracellularly applied electrical fields on the excitability of spinal motoneurons with the aim of reducing the increased motoneuronal excitability after spinal cord injury (SCI). Our results suggest that electrical fields could suppress the excitability of motoneurons and reduce their firing rate significantly by modulating the magnitude of their dendritic PIC. This effect was achieved at different field directions, intensities, and polarities. The reduction in motoneuronal firing rate resulted from the reduction in the magnitude of the dendritic PIC reaching the soma by the effect of the applied electrical field. This reduction in PIC was attributed to the dendritic field-induced differential polarization and the nonlinear current-voltage relationship of the dendritic PIC-mediating channels. Because of the location of the motoneuronal somata and initial segment with respect to the dendrites, these structures were minimally polarized by the applied field compared with the extended dendrites. In conclusion, electrical fields could be used for suppressing the hyperexcitability of spinal motoneurons after SCI and reducing the level of spasticity.

Modulation of motoneuronal firing behavior after spinal cord injury using intraspinal microstimulation current pulses: a modeling study.

We simulated the effects of delivering focal electrical stimuli to the central nervous system to modulate the firing rate of neurons and alleviate motor disorders. Application of these stimuli to the spinal cord to reduce the increased excitability of motoneurons and resulting spasticity after spinal cord injury (SCI) was examined by means of a morphologically detailed computer model of a spinal motoneuron. High-frequency sinusoidal and rectangular pulses as well as biphasic charge-balanced and charge-imbalanced pulses were examined. Our results suggest that suprathreshold high-frequency sinusoidal or rectangular current pulses could inactivate the Na+ channels in the soma and initial segment, and block action potentials from propagating through the axon. Subthreshold biphasic charge-imbalanced pulses reduced the motoneuronal firing rate significantly (up to approximately 25% reduction). The reduction in firing rate was achieved through stimulation-induced hyperpolarization generated in the first node of Ranvier. Because of their low net DC current, these pulses could be tolerated safely by the tissue. To deliver charge-imbalanced pulses with the lowest net DC current and induce the largest reduction in motoneuronal firing rate, we studied the effect of various charge-imbalanced pulse parameters. Short pulse durations were found to induce the largest reduction in firing rate for the same net DC level. Subthreshold high-frequency sinusoidal and rectangular current pulses and low-frequency biphasic charge-balanced pulses, on the other hand, were ineffective in reducing the motoneuronal firing rate. In conclusion, the proposed electrical stimulation paradigms could provide potential rehabilitation interventions for suppressing the excitability of neurons to reduce the severity of motor disorders after injury to the central nervous system.

GABAB receptor: a site of therapeutic benefit.

Although the presence of functional GABAB receptors in mammalian brain has been known for more than 20 years, there is still only one therapeutic agent in use, baclofen, which mediates its effects directly via this receptor. However, activation of this receptor can produce numerous effects that might be amenable to drug development. Evidence from preclinical studies also suggests that antagonism of the GABAB receptor produces beneficial clinical effects.

Energy cost and physiological reactions to effort during activation of paraplegics by functional electrical stimulation.

In this study, the aim was to evaluate the influence on the cardiopulmonary system of muscular contractions of the paralyzed limbs in paraplegia, activated by FES during treatment, and the energy cost of standing and walking while using FES as an orthotic aid. Three traumatic spastic paraplegics were selected for the measurements. At the end of a 6 month training program heart rate and oxygen consumption of the patients were evaluated as follows: at rest; following 30 minutes of FES in the sitting position; following 15 minutes of standing; and during ambulation. Lactic acid level during maximal effort was evaluated as well. The results indicated a low energy cost of FES in the sitting position and during usage of FES as an orthotic device for standing, confirming the beneficial effect of FES for spastic paraplegics. However, effort invested during ambulation by means of FES was found exhaustive and FES is therefore advisable for young subjects mainly.