Inhibitory role of lentivirus-mediated aquaporin-4 gene silencing in the formation of glial scar in a rat model of traumatic brain injury.

Traumatic brain injury (TBI), an acute degenerative pathology of the central nervous system, is a leading cause of death and disability. As the glial scar is a mechanical barrier to nerve regeneration, inhibitory molecules in the forming scar and methods to overcome them have suggested molecular modification strategies to allow neuronal growth and functional regeneration. Herein, we aim to investigate the effects of aquaporin-4 (AQP4) gene silencing on the glial scar formation after TBI by establishing rat models. After modeling, TBI rats were transfected with AQP4 small hairpin RNA [shRNA] (AQP4 gene silencing by lentiviral vector-delivered shRNA) and empty vectors, respectively. Neurological functions of the rats were evaluated after TBI. The hematoxylin and eosin staining was conducted to observe histomorphological changes in rat brain tissues. The messenger RNA (mRNA) and protein expressions of glial fibrillary acidic protein (GFAP), vimentin, fibronectin, laminin, and AQP4 were measured by reverse transcription-quantitative polymerase chain reaction and Western blot analysis. The ratio of positive expression area was calculated, and the glial scar was observed by immunohistochemistry. At the 7th, 14th, and 28th days after TBI, TBI rats treated with AQP4 shRNA showed improved neurological function and lessened histomorphological changes. AQP4 gene silencing mediated by lentivirus decreased the mRNA and protein expressions of GFAP, vimentin, fibronectin, and laminin, the number of positive cells, the ratio of positive expression area, and the glial scar. Our study demonstrates that lentivirus-mediated AQP4 gene silencing could inhibit the formation of glial scar after TBI, which is beneficial to the recovery of neurological function.

GABA-ergic interneurons involved in transcallosal inhibition of the visual cortices in vivo in mice.

In the current study we investigated the role of the corpus callosum, particularly the gamma-aminobutyric acid-ergic (GABAergic) projection neurons involved in interhemispheric inhibition (IHI). In order to explore IHI in primary visual cortices, we adopted a protocol whereby we performed a direct current lesion of the unilateral primary visual cortex with or without posterior callosotomy, and used two-photon Ca(2+)in vivo imaging on the opposite unaffected region to detect neural activities in mice. Following this procedure, the numbers of vesicular GABAergic transporters (VGATs) and GABAergic interneurons in the unaffected primary cortex were determined using immunofluorescence staining. Results indicated that following unilateral visual cortical lesioning without callosotomy, the neuronal Ca(2+) activities in the opposite side were significantly increased. However, the neuronal activities of the unaffected visual cortex in animals with unilateral cortical lesion with callosotomy were not significantly different. Additionally, there was no significant difference in the numbers of GABAergic interneurons in the unaffected region between each group, while the number of VGATs in the unaffected region was significantly decreased following unilateral visual cortical lesion without callosotomy, which was unchanged once with callosotomy. Finally, callosotomy alone without cortical lesioning produced no change in neuronal activities, the number of GABAergic interneurons or VGATs. Our results demonstrate that IHI between the homologous primary visual cortices occurs via the corpus callosum, and further indicate the important involvement of long-range GABAergic interneurons in transcallosal inhibition.

Allopurinol protects against ischemic insults in a mouse model of cortical microinfarction.

Microinfarcts are common in patients with cognitive decline and dementia. Allopurinol (ALLO), a xanthine oxidase (XO) enzyme inhibitor, has been found to reduce proinflammatory molecules and oxidative stress in the vasculature. We here examined the effect of pre-treatment with allopurinol on the cortical microinfarction. C57BL/6J mice were subjected to a permanent single penetrating arteriole occlusion induced by two-photon laser irradiation. Infarction volume, the activation of glial cells and nitrosative stress in the ischemic brain was assessed using immunohistochemistry. Pre-treatment with ALLO achieved 42% reduction of infarct volume and significantly reduced microglia infiltration, astrocyte proliferation and nitrosative stress in the ischemic brain. These data indicate that ALLO protects against microinfarcts possibly through inhibition of nitrosative stress and attenuation of microglia infiltration as well as astrocytes reactivation.

The high frequency and clinical feature of seronegative myasthenia gravis in Southern China.

Anti-acetylcholine receptor antibodies (anti-AChR-Ab) are responsible for the failure of neuromuscular junction in myasthenia gravis (MG). Some anti-AChR-Ab-seronegative MG patients have anti-muscle-specific tyrosine kinase antibodies (anti-MuSk-Ab). Here, the anti-AChR-Ab was tested in 250 MG outpatients from Southern China. While anti-MuSk-Ab was tested in 66 patients who had no anti-AChR-Ab in blood serum, but none of them was positive. The antibodies were measured by a radioimmunoprecipitation assay. The frequency of anti-AChR-Ab was 51.2 %. The percentage of anti-AChR-Ab in ocular type was lower than generalized type (44.9 vs. 66.2 %, P = 0.002). Seronegative MG was characterized by a lower percentage of thymoma than seropositive patients (P = 0.013). It seemed to be less severe in seronegative MG than seropositive MG in these 250 patients. In ocular type, seronegative MG mainly manifesting blepharoptosis but seldom diplopia or eyeball fixation related to ocular movement disability (P = 0.016). While in generalized type, seronegative MG was characterized by a lower percentage of bulbar muscle involvements than seropositive patients (P = 0.005). Logistic regression analysis revealed that bulbar weakness was affected by the existence of anti-AChR antibodies (OR = 3.524, P = 0.015). Besides, seronegative MG tended to be characterized by a lower percentage of neck extensor involvement, but this did not reach significance. The percentage of anti-AChR antibodies was much lower than other countries. Seronegative MG has characteristic clinical features that are different from features of the remaining seropositive MG. This emphasises the predictive value of anti-AChR antibodies analysis in MG patients.

[Efficacy and safety of low-dose cyclophosphamide plus corticosteroids for type I/II myasthenia gravis].

OBJECTIVE: To evaluate the efficacy and safety of low-dose cyclophosphamide plus corticosteroids for type I/II myasthenia gravis (MG). METHODS: This trial was prospective, non-random and open-labeled. We selected 160 patients with steroid-insensitive MG from January 1999 to October 2011. Each patient received an oral dose of prednisone 0.5 mg×kg(-1)×d(-1), cyclophosphamide 8 mg×kg(-1)×time(-1) once weekly intravenously and oral pyridostigmine 36 mg×kg(-1)×d(-1). The efficacies were assessed by absolute and relative MG scores. The clinical treatment cycle was 30 weeks. RESULTS: (1) Among them, 74/106 type I MG patients (69.9%) reached clinical relative scores ≥ 95% in 30 weeks. The total dose of cyclophosphamide was 12 g. And 35/54 type II MG patients (64.8%) reached clinical relative scores ≥ 95% in 30 weeks. No statistically significant difference existed between two groups (P = 0.521). (2) All patients had various degrees of improvement in 30 weeks. The difference was statistically significant by Mann-Whitney U test (P ≤ 0.05). (3) There were minor side effects in these all patients. (4) When the total dose of cyclophosphamide reached 4 g, the cure rate in type I patients was higher than that of in type II patients (P = 0.000). When the total dose of cyclophosphamide reached 12 g, the cure rate in type II patients was higher than that of in type I patients (P = 0.001). (5) The cure dose of cyclophosphamide was 4-8 g in 58.4% of type I patients versus 8 - 12 g in 61.1% of type II patients. CONCLUSION: The combined treatment of low-dose cyclophosphamide and corticosteroids in glucocorticoid-insensitive type MG (type I/II) is both effective and safe. And the sensitivity to cyclophosphamide varies for different clinical types. It is higher in type I than type II patients.

[Randomized controlled clinical trial of middle-dose cyclophosphamide plus methylprednisolone for myasthenia gravis patients in crisis].

OBJECTIVE: To evaluate the efficacy and safety of middle-dose cyclophosphamide plus methylprednisolone for myasthenia gravis (MG) patients in crisis. METHODS: For this prospective, open, parallel, randomized controlled trial, we recruited a total of 156 MG patients in crisis from January 1999 to October 2011 at Department of Neurology, First Affiliated Hospital, Sun Yat-sen University. They were divided into two groups of cyclophosphamide and control (n = 78 each). In the cyclophosphamide group, each received methylprednisolone 500 mg/d for 3 days, then tapered to 250 mg/d and tapered half every 3 days until 62.5 mg/d. Afterward an oral dose of prednisone was prescribed at 30 mg/d until the end of the trial. At the same time, an intravenous injection of cyclophosphamide was offered at 0.4 g/d for 3 days and then 0.4 g/d every 3 days. In the control group, each received methylprednisolone alone. And the efficacies were assessed by absolute and relative MG scores. RESULTS: (1) There were 54 (69.2%) patients off-ventilation in 3 days in the cyclophosphamide group versus 36 (46.2%) patients in 8 - 14 days in the control group. Notable statistical significance existed between two groups (P = 0.000). (2) More than half of the patients in cyclophosphamide group with extremity weakness (n = 44, 56%) and dysphagia (n = 47, 60.3%) significantly improved in 10 - 14 days versus 28 days in the control group. Notable statistical significance existed between two groups (P = 0.000). (3) In the cyclophosphamide group, dyspnea disappeared in 54 (69.2%) patients when the dose reached 1.2 g. The recovery of dysphagia (n = 47, 60.3%) and extremity weakness (n = 44, 56.4%) occurred in more than half of the patients when the dose reached 2.8 g. Notable statistical significance existed among three groups (P = 0.000). (4) During the treatment period, there were 17 cases (21.8%) with pulmonary infection in the cyclophosphamide group versus 53 cases (67.9%) in the control group. Notable statistical significance existed between two groups (P = 0.000). (5) Brief and minor side effects appeared in the patients of the cyclophosphamide group. CONCLUSION: (1) The combined treatment of middle-dose cyclophosphamide and methylprednisolone for MG patients in crisis is both effective and safe. (2) When combined with methylprednisolone, 90% of patients with MG crisis are successfully off-ventilation when the dose of cyclophosphamide reaches 1.6 g.

[Response to thymectomy and analysis of influencing factors in the treatment of children with myasthenia gravis].

OBJECTIVE: To evaluate the efficacy of thymectomy and relevant influencing factors in the treatment of children with myasthenia gravis through a long-term follow-up. METHODS: The clinical records of 59 patients undergoing expanded thymectomy for the treatment of myasthenia gravis (MG) between January 2003 and August 2009 were reviewed retrospectively. Their postoperative outcomes were categorized into complete stable remission (CSR), pharmacological remission (PR), improvement, no change and deterioration (including mortality). RESULTS: During a median follow-up period of 35 months, none of them died or deteriorated clinically among 53 patients with a postoperative follow-up. The overall remission rate was 69.8% and the effective rate 90.6%. No symptomatic relapse occurred among 16 patients in CSR. None of the ocular patients progressed to generalized MG while 16 thymectomized generalized MG developed from ocular MG. Both univariate and logistic regression analyses revealed that the preoperative duration of illness influenced the surgical curative effect (P < 0.05). Survival analysis indicated that the rates of overall remission were 56% or 88% at 24 months and 42% or 75% at 48 months among ocular MG and generalized MG respectively. According to Log-rank analysis, no difference in remission existed between two types of MG. CONCLUSION: Thymectomy is an effective and safe treatment in selected MG children, especially in those with a shorter illness duration.

[Clinical study and case follow-up of 61 cases of familial myasthenia gravis in 28 families].

OBJECTIVE: To explore the clinical features and prognosis of familial myasthenia gravis (FMG). METHODS: The clinical data were collected and analyzed for 61 FMG patients from 28 families from February 1998 to March 2009 at the department of neurology, First Affiliated Hospital, Sun Yat-sen University. And they were compared with 257 cases of sporadic myasthenia gravis (MG) by case-control method. Age at onset, gender, Osserman clinical classification, diagnosis, treatment, thymus imaging examination, prognosis and other family members were measured retrospectively. RESULTS: (1) The proportion of FMG in Southern China was 2.03% and it was lower than the other countries. (2) Onset age was younger in FMG than in sporadic MG. Juvenile MG was more common in FMG than in sporadic MG (70.5% vs 52.1%, P = 0.009). (3) Ocular MG was more common in FMG than in sporadic MG (83.6% vs 62.7%, P = 0.002). (4) There was a higher proportion in FMG with thymic hyperplasia than in sporadic MG (98.3% vs 83.7%, P = 0.014). (5) Better prognosis was found in FMG than in sporadic MG. FMG patients had a higher proportion of completely stable remission than sporadic MG (24.5% vs 11.7%, P = 0.009). (6) The so-called "consistency of the same disease" could only be found in families with pure ocular diseases. If the patients with general type MG were present in the family, their pathogenetic conditions and prognosis might vary greatly. CONCLUSION: There characteristics of FMG in south China are different from those of other countries. Their exact inheritance patterns await further explorations.

A retrospective review of 15 patients with familial myasthenia gravis over a period of 25 years.

We observed, during a 25-year period, 15 patients from 6 families with autoimmune myasthenia gravis (all Chinese Han from Guangdong Province) referred to our department. Their mean onset age was 13.4 years (range 2-25 years) with 10 patients with juvenile onset. The female:male ratio was 3:2. Acetylcholine receptors antibody titers were increased in 11 patients (range 1.62-19.8 nmol/L). Thymectomy was performed in six patients, who received corticosteroids /immune inhibitor plus pyridostigmine treatments after surgery. The other patients were placed on therapy with azathioprine, cyclophosphamide, corticosteroids and acetylcholinesterase inhibitors. All patients responded well to immunosuppressants, and psychiatric symptoms were observed only in one patient who received a high dose of corticosteroids. Patients with generalized type in the same family had different presentations with variable prognosis. HLA-A 0207 was found in 9 patients (9/15), HLA-B 4601 in 11 patients (11/15), and HLA-DRB1 0901 in 12 patients (12/15). When compared to familial autoimmune myasthenia gravis in other countries, we observed peculiar characteristics of Chinese populations, such as the within-family consistency was only found in families with ocular MG type (50% of all MG families), while the pathogenetic conditions and the prognoses of the generalized MG patients may differ greatly within the same family. These findings may shed new light on the genetic predisposition and the origin of immune abnormalities of MG patients.

[Study of incidence and correlation factors of depression, anxiety and insomnia in patients with myasthenia gravis].

OBJECTIVE: To investigate the incidence rate and correlation factors of depression, anxiety and insomnia in patients with myasthenia gravis (MG). METHODS: A total of 161 MG patients were assessed and graded with HAMD, HAMA, PSQI, QMG, ADL and a self-made scale chart. And the correlation factors were analyzed by Logistic stepwise regression. RESULTS: The prevalence of depression, anxiety and insomnia was 58.3%, 45.3% and 39.1% respectively. The correlation factors with significant influences on MG were as follows: depression with age, physical weakness, score of QMG, life scale grading; anxiety with experience-sharing; insomnia age, dyspnea, thymoma, physical status at 1 month post-operation, prednisone dose and score of QMG. CONCLUSION: Nearly one half of the MG patients suffer from affective disorders to different degrees. And an analysis of its correlation factors provides references to prevent and treat the affective disorders concurrently with MG.