10.3969/j.issn.1000-8179.2013.12.016

Objective To explore the mechanism of neurotoxic effects of neuron nitric oxide synthase(nNOS)and inducible nitric oxide synthase(iNOS),and the therapeutic effects of 7-nitroindazole(7-NI)and aminoguanidine(AG),in traumatic brain injury(TBI)rats.Methods Two hundred and fifty adult male Wistar rats were randomly divided into 5 groups:sham operation group,traumatic group,AG group,7-NI group and AG+7-NI group.Animals in each group were divided into 8 subgroups according to the time after trauma(1,3,6,12 hours and 1,3,7,14 days).Severe diffused brain injury model was made with Marmarou method.Expressions of nNOS and iNOS in hippocampus CA1 region were determined by immunohistochemistry,nerve cells apoptosis in hippocampus CA1 region was observed by TUNEL methods,and the relationship between apoptosis and NOS was observed by double staining.Results In trauma group,the expression of nNOS in hippocampus CA1 region peaked at 6h post-trauma,the expression of iNOS and apoptosis of nerve cells in hippocampus CA1 region both peaked at 3d post-trauma,while the apoptosis was alleviated in AG group,7-NI group and AG+7-NI group.The number of both TUNEL staining and nNOS immunostaining positive cells increased at 6h post-trauma in trauma group,significantly higher than that in 7-NI group.The number of both TUNEL staining and iNOS immunostaining positive cells increased at 3d post-trauma in trauma group,significantly higher than that in AG group.Conclusions The over-expression of nNOS and iNOS has toxic effects to neural tissues of brain,serves as one of the factors inducing nerve cell apoptosis in hippocampus CA1 region.7-NI and AG can inhibit the expression of nNOS and iNOS,reduce the nerve cell apoptosis,and play an important role in neuroprotective effect.

Objective To explore the influence of injury severity on transplantation of embryonic neural stem cells (NSCs) after traumatic brain injury (TBI).Methods The NSCs were isolated from the hippocampus of fetal rats aged at from 12-14 days.The cells were cultured and proliferated in the serum-free medium and identified in vitro.The animals received transplants in the bilateral hippocampal areas at day 3 following mild or moderate TBI separately.Conventional histology,TUNEL and immunohistology were examined to detect BrdU,NSE,GFAP,GalC,NGF and BDNF at day 14 post-implantation.Results BrdU-labeled positive cells in the bilateral hippocampus in the mild TBI group were more than those in the moderate TBI group at day 14 post-implantation.Significant differentiation of the astrocytes recognized as GFAP positive cells in the bilateral hippocampus was found at day 14 post-implantation.The expression of NGF and BDNF proteins was increased following TBI,the most evident in the mild TBI group.Conclusion The influence of injury severity on transplantation may be associated with the change of the microenvironment after TBI.

Objective To observe the efficacy of pulsed dual-wavelength 595 and 1064 nm laser in treating ulcerated infantile hemangioma.Methods Lesions from 40 patients were treated with the laser.The distribution of lesion sites was in buttuck and perianal area (13 patients),vulvar lips (7),groin and inside thigh (5),scrotum (4),neck (3),scalp (2),lip (2),trunk and limbs (4).All lesions including ulcer and non-ulcer were conducted with 595 nm plused dye leaser at the first therapy.Later,595 nm pulsed dye leaser was continued or performed with Group 1 or Group 2 parameter of 595 and 1064 nm multiplex mode until the treatment was finished according to the thickness of the lesion.The frequency of treatment was one to six times.The interval of the treatment was four weeks.Results The ulcer from 39 lesions healed gradually in 1 to 2 weeks (average 9.5 days) after the first treatment.These ulcer lesions were healed four weeks after the first time of therapy.The recovery rate of ulcerated lesion was 97.5 ％.For the whole lesion,after 1 to 6 treatments,13 lesions showed excellent result,19 lesions showed good result,and the effective rate was 80 ％.Pain relieved 2 to 7days (average 3.5 days) after the first laser therapy in 35 patients who accompanied with pain.Pain score decreased from 1.875 to 0.125 before and after treatment,with significant difference(P＜0.05).The tolerance of the treatment was good and no side effect was observed.Conclusions 595and 1064 nm dual-wavelength laser is a safe and effective tool for treating ulcerated infantile hemangioma.

Objective To study the mechanism of dermal fibroblasts as a feeder layer to support the growth of human keratinocytes. Methods Human dermis fibroblasts were isolated and cultured and then treated with mitomycin-C. The expression of type Ⅰand type Ⅲ precollagen mRNA and relevant protein in feeder layer were examined by RT-PCR and Immunohistochemistry. KCs were cultured both on FB and NIH3T3 feed layer as control, the adhering numbers and the time of fusion were recorded. Results RT-PCR showed an increase of type Ⅰprecollagen mRNA in FB feeder layer as compared with that of normal fibroblasts (P

Objective To optimize human acellular dermal matrix(ADM) and evaluate its biological characters. Methods Human skin was treated with hypertonic saline followed by NaOH maceration(group A), hypertonic saline followed by sodium dodecyl sulfate (SDS) detergent(group B) or Dispase Ⅱ followed by Triton X-100(group C), the resulting ADM were sectioned, and then were stained by special immunohistochemistry method. The cytotoxicity of them were evaluated by methyl thiazolyl tetrazolium (MTT) colorimetry and then cell compatibility was analyzed by cell culture;The optimized ADM resulted was choosen for use. Fibrablasts(FBs)were transfected with adenovirus vector encoding green fluorescent protein gene(Ad-GFP)and the growth of them on the optimized ADM was observed by fluorescent microscopy. Results Collagen and elastic fibers can still be observed in three kinds of ADM. The cells in dermis can be disintegrated both in group A and C, but not in group B. The cytotoxicity scores of the ADM prepared in group A and B were grade 0 or grade 1, while that of group C was more than grade 1.The ADM prepared by NaCl-NaOH maceration had good biocompatibility. There was statistical difference in adhering number of NIH3T3 cells in group A and B. NIH3T3 cells grew well in group A and the resulted ADM was optimized. FBs transfected with Ad-GFP grew well in the optimized ADM. Conclusion The ADM prepared by NaCl-NaOH maceration was a good tissue engineering biomaterial with a little cytotoxicity and rich in resouce.

Objective To study the mechanism of interaction between neuronal nitric oxide syn-thase (nNOS) and inducible nitric oxide synthase (iNOS) following traumatic brain injury (TBI) in rats. Methods A total of 250 male Wistar rats were randomly divided into five groups, ie, sham oper-ation group, trauma group, 7-nitroindazole (7-NI) treatment group, aminoguanidine (AG) treatment group and combined AG and 7-NI treatment group. Severe closed TBI was made by using Marmarou meth-od. Protein expressions of nNOS and iNOS in hippocampus CAI were detected by means of immunohisto-chemical staining at 1,3, 6, 12 hours and at days 1,3, 7 and 14 after TBI. Results The expression of nNOS reached a peak at 6 hour after injury in all groups, with no statistical difference between groups (P > 0. 05), when there was no statistical difference between 7-NI treatment group and trauma group (P > 0. 05) but statistical difference in AG treatment group and combined AG and 7-NI treatment group compared with trauma group at 12 hours after TBI (P <0.05). The expression of iNOS reached maximal level at day 3 after TBI, with lower level in 7-NI group, AG treatment group and combined AG and 7-NI treatment group compared with trauma group (P < 0.05). Conclusions After TBI, nNOS interacts with iNOS by means of the feedback of nitric oxide. The enhanced expression of nNOS is initial factor for increase of iNOS expression, which can down regulate the expression of iNOS.

Objective To determine the clinical value of pulsed dual-wavelength 595 and 1064 nm laser in treating superficial infantile hemangioma. Methods The treatments were conducted in 260 patients with 270 lesions. The location of the lesions were 41 cases in scalp, 88 in face and neck, 79 in trunk and 62 in arms and legs. All lesions were treated several times with 595 nm (Group 1) and/or 1064 nm (Group 4) multiplex mode until the treatment was finished. The frequency of treatment was one to six times. The interval of the treatment was four weeks. Results The total effective rate was 95.56 %, with atrophic scar (8.15 %) but without severe ulcer. No significance was found as compared Group 1 parameter with Group 4 in formation of atrophic scars (x2 = 1. 870, P＞0.05). Significant difference in the incidence of atrophic scars was found among different location of lesions (x2 = 15. 743, P＜0. 01) : lesion in face had the highest probability. No obvious difference was found between different location of lesion and the frequence of treatment (x2 = 21.164, P＞0.05), and so did with the efficacy (x2 = 11. 597, P＞0.05). The size and the thickness of lesion had significant difference in the time of treatment (x2 = 58. 171, P＜0. 01 and x2 = 11. 583, P＜0.05, respectively).Conclusions 595nm/1064 nm dual-wavelength laser is a safe and effective tool for treating superficial infantile hemangioma. Choice of treatment parameter in the mode depends on the features of vascular lesions.

Objective To observe expression of autophagy-related proteins LC-3 and Beclin-1 in alveolar macrophages in the silicosis model of rats , and to investigate the molecular mechanisms of silicosis formation from cells autophagy perspective .Methods Fifty adult male SD rats were randomly divided into control and model groups , 25 rats for each group .The silicosis model was made by one-time infusion of silica dust suspension through the trachea without exposed.The rats were sacrificed on the 1st, 3rd, 7th, 14th or 28th day after the modeling .Alveolar macrophages were obtained from bronchoalveolar lavage and used for subsequent research after culture and purification .Morphological characteristics of alveolar macrophages were observed by HE staining and transmission electron microscope ;The expression of LC-3 and Beclin-1 was detected by means of immunocytochemistry and Western blotting in each group .Results Compared with the control group , alveolar macrophages of the model group had larger volume and abundant cytoplasm , the phagocytic silica dust particles were observed in some cells , and autophagosomes were detected by transmission electron microscope .The expressions of LC-3 and Beclin-1 were increased at all time points in the model group ( P<0.05 ) .Both LC-3 and Beclin-1 began to increase at the 1st day.As the extension of time the expression gradually enhanced , peaked at the 14th day(P<0.05), and decreased at the 28th day, but higher than the basal expression .Conclusion Autophagy is activated in alveolar macrophages of the silicosis model , and alveolar macrophages autophagy is involved in the pathological process of silicosis in the rat .

Objective To investigate the clinical therapeutic effect of mild hypothermia in elderly patients with severe traumatic brain injury.Methods 72 cases of elderly patients with severe traumatic brain injury(GCS ≤ 8) were divided into mild hypothermia therapy group(36 patients)and control group(36 patients) according to the random number table method.Mild hypothermia therapy group received mild hypothermia treatment while control group received normal treatment.The clinical prognosis was analyzed between the two groups.Results After 24h treatment,both mild hypothermia therapy group and control group intracranial pressure began to rise.But the intracranial pressure of the mild hypothermia therapy group(24 h:(13.0±4.5)mmHg,3 d:(16.6±4.0) mmHg,5 d:(19.9±3.9) mmHg,1 mmHg=0.133 kPa) were significantly lower than those of the control group (24 h:(16.6± 3.8) mmHg,3 d:(20.4±4.8) mmHg;5 d:(24.1 ± 6.2) mmHg),and the difference was statistically significant (t=2.225,2.260,2.192,P=0.035,0.033,0.039).The rate of good recovery to the control group and the mild hypothermia therapy group were 22.22％ and 47.22％ respectively while the mortality were 30.56％ and 13.89％ respectively,and the differences were statistically significant (x2 =4.936,5.675,P=0.047,0.035).Conclusion Mild hypothermia treatment can inhibit the increase of intracranial pressure and reduce disability rate and mortality in elderly patients with severe traumatic brain injury,which can increase the survival rate and improve the long-term prognosis.