Effects of intracerebral administration of IL-1beta on reactive behaviors of astrocytes and macrophages in the injured brain of newborn rats

Newborn male rats were subjected to a mechanical lesion of the left cerebral hemisphere. Thereafter, a single dose 5 or 500 units (U) of recombinant rat interleukin-1beta (IL-1beta) was injected into the lesion cavity. One or 2 days after the injury, the rats were injected with 3H-thymidine to label dividing cells. Brain sections were subjected to GFAP immunocytochemistry or BSI-B4 lectin histochemistry to visualise astrocytes or macrophages, respectively. Autoradiography was used to detect cells proliferating within the region of injury in the immunocytochemically stained brain sections. The strongest mitogenic effect of IL-1beta on astrocytes (labeling index) was observed on day 1 after injury while a dose-dependent increase in their GFAP-immunoreactivity occurred on day 2. At 500U dose, IL-1beta significantly reduced infiltration of macrophages on posttraumatic days 1 and 2 but did not influence their proliferation. Thus, effects of IL-1beta on the occurrence of macrophages were opposite to those on the GFAP-immunoreactivity of astrocytes and their proliferation. It appears to suggest existence of different mechanisms controlling reactive behaviors of the two cell populations.     

A comparison of the proliferative activity of astrocytes and astrocyte-like cells expressing vimentin in the injured mouse cerebral hemisphere.

After an unilateral injury of the cerebral hemisphere, 28 nice were injected with 3H thymidine at different intervals following the injury. Thereafter, the distribution of autographically labelled astrocytes expressing glial fibrillary protein (GFAP) and astrocyte-like cells expressing vimentin were recorded within the region of injury. Proliferative activity of the two cell types started at the same time, i.e. 24 h. after injury, reached its maximum on day 4, and returned to normal level after the 8th posttraumatic day. However, on day 4 the number of proliferating GFAP-positive astrocytes was about 50% higher than that of vimentin-positive cells. This was regarded as a proof of the concept that a significant number of astrocytes did not express vimentin during its mitotic cycle. Those facts were considered as an evidence against the hypothesis that a reactive astrocyte division induces a two-stage increase in the cytoskeletal proteins level with the elevated synthesis of vimentin preceding that of GFAP.     

Regulated expression of pancreatic triglyceride lipase after rat traumatic brain injury

Pancreatic triglyceride lipase (PTL), an enzyme of digestive system, plays very important roles in the digestion and absorption of lipids. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of PTL during traumatic brain injury (TBI). Western blot and RT–PCR analysis revealed that PTL was present in normal rat brain cortex. It gradually increased, reached a peak at the 3rd day after TBI, and then decreased. Double immunofluorescence staining showed that PTL was co-expressed with neuron, but had a few colocalizations in astrocytes. When TBI occurred in the rat cortex, the expression of PTL gradually increased, reached the peak at the 3rd day after TBI, and then decreased. Importantly, more PTL was colocalized with astrocytes, which is positive for proliferating cell nuclear antigen (PCNA). In addition, Western blot detection showed that the 3rd day post injury was not only the proliferation peak indicated by the elevated expression of PCNA, glial fibrillary acidic protein (GFAP) and cyclin D1, but also the apoptotic peak implied by the alteration of caspase-3 and bcl-2. These data suggested that PTL may be involved in the pathophysiology of TBI and PTL may be complicated after injury, more PTL was colocalized with astrocytes. Importantly, injury-induced expression of PTL was colabelled by proliferating cell nuclear antigen (proliferating cells marker), and the western blot for GFAP, PCNA and cyclin D1, showed that 3 days post injury was the proliferation peak, in coincidence to it, the protein level change of caspase-3 and bcl-2 revealed that the stage was peak of apoptotic too. These data suggested that PTL may be involved in the pathophysiology of TBI and that PTL may be implicated in the proliferation of astrocytes and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of PTL after brain injury.     

大鼠脑缺血再灌注后神经元和星形胶质细胞凋亡的比较研究

目的比较研究大鼠局灶性脑缺血再灌注后神经元和星形胶质细胞的凋亡规律。方法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,在缺血再灌注后1、3、7、14d断头取脑,应用流式细胞分选技术和原位末端标记法分别检测各组MCAO后不同时期神经元和星形胶质细胞凋亡情况。结果局灶性脑缺血再灌注后,海马区星形胶质细胞凋亡数量超过神经元,其凋亡以再灌注3d最为显著,而神经元则以7d最为显著;而皮层区神经元凋亡数量超过星形胶质细胞,两种细胞凋亡均在再灌注后7d达高峰。结论脑缺血再灌注后,皮层和海马区的神经元及星形胶质细胞均可发生凋亡,海马区星形胶质细胞比皮层区更易凋亡,而皮层区神经元比海马区更易凋亡。     

电针对脊髓损伤星形胶质细胞增生及其NGF表达的影响

目的研究脊髓损伤后电针治疗对星形胶质细胞增生及其内源性神经生长因子(nerve growth factor,NGF)表达的影响.方法选用成年雌性Wistar大鼠,随机分为3组.A组为正常对照组,B组、C组为下胸段脊髓不完全损伤.B组损伤后不治疗,C组损伤后给予督脉电针治疗.损伤后3 d、1 、2或4周应用免疫组化染色分别观察损伤脊髓胶质原纤维酸性蛋白(glial fibroblast acid protein,GFAP)和NGF表达的变化.结果 B组术后3 d,GFAP阳性细胞明显增多, 2周后开始减少,4周时仍有较多的阳性细胞;C组GFAP阳性细胞明显少于B组,1周时达高峰.脊髓损伤后NGF表达呈逐渐增加的趋势.C组NGF的表达明显高于B组,且一直保持在较高水平.NGF阳性细胞大部分与GFAP阳性细胞形态相似.结论电针治疗能减少星形胶质细胞增生,促进内源性NGF的合成,从而创造了有利于神经再生的微环境.     

LIN28 Expression in Rat Spinal Cord After Injury

LIN28, an RNA-binding protein, is known to be involved in the regulation of many cellular processes, such as embryonic stem cell proliferation, cell fate succession, developmental timing, and oncogenesis. However, its expression and function in central nervous system still unclear. In this study, we performed an acute spinal cord contusion injury (SCI) model in adult rats and investigated the dynamic changes of LIN28 expression in spinal cord. Western blot and immunohistochemistry analysis revealed that LIN28 was present in normal spinal cord. It gradually increased, reached a peak at 3 day, and then nearly declined to the basal level at 14 days after SCI. Double immunofluorescence staining showed that LIN28 immunoreactivity was found in neurons, astrocytes and a handful of microglia. Interestingly, LIN28 expression was increased predominantly in astrocytes but not in neurons. Moreover, the colocalization of LIN28 and proliferating cell nuclear antigen was detected after injury. Western blot showed that LIN28 participated in lipopolysaccharide (LPS) induced astrocytes inflammatory responses by NF-κB signaling pathway. These results suggested that LIN28 may be involved in the pathologic process of SCI, and further research is needed to have a good understanding of its function and mechanism.     

Upregulation of p21-activated Kinase 6 in rat brain cortex after traumatic brain injury

p21-activated Kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. PAK kinases are well-known regulators of a wide variety of cellular functions, including regulation of cytoskeleton rearrangement, cell survival, apoptosis and the mitogen-activated protein kinase signaling pathway. To elucidate the expressions and possible functions of PAK6 in central nervous system (CNS) lesion and repair, we performed a traumatic brain injury (TBI) model in adult rats. Western blot analysis revealed that PAK6 level significantly increased at day 3 after damage, and then declined during the following days. Besides, double immunofluorescence staining showed PAK6 was primarily expressed in the neurons and a few of glial cells in the normal group. While after injury, the expression of PAK6 was increased significantly in the astrocytes and neurons, and the astrocytes had largely proliferated. We also examined the expression of proliferating cell nuclear antigen (PCNA) whose change was correlated with the expression of PAK6. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many PAK6-expressing cells at day 3 after injury. In addition, injury-induced expression of PAK6 was co-labeled by active caspase-3 during neuronal apoptosis after injury. Collectively, we hypothesized PAK6 may play important roles in CNS pathophysiology after TBI and further research is needed to have a good understanding of its function and mechanism.     

Spatiotemporal Pattern of RNA-Binding Motif Protein 3 Expression After Spinal Cord Injury in Rats

RNA-binding motif protein 3 (RBM3) belongs to a very small group of cold inducible proteins with anti-apoptotic and proliferative functions. To elucidate the expression and possible function of RBM3 in central nervous system (CNS) lesion and repair, we performed a spinal cord injury (SCI) model in adult rats. Western blot analysis revealed that RBM3 level significantly increased at 1 day after damage, and then declined during the following days. Immunohistochemistry further confirmed that RBM3 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased at 1 day after SCI. Besides, double immunofluorescence staining showed RBM3 was primarily expressed in the neurons and a few of astrocytes in the normal group. While after injury, the expression of RBM3 increased both in neurons and astrocytes at 1 day. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase-3 in injured spinal cords by western blot. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many RBM3-expressing cells and rare caspase-3 was observed in RBM3-expressing cells at 1 day after injury. Our data suggested that RBM3 might play important roles in CNS pathophysiology after SCI.     

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP(+) astrocytes (21% of FABP7(+) cells) and NG2(+) oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7(+)/NG2(+) cells, while there was a significant increase in FABP7(+)/GFAP(+) cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU(+) astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.     

Experimental Investigation of HGF Inhibiting Glial Scar In Vitro

To study the inhibitory effect of Hepatocyte growth factor (HGF) on the responsive hyperplasia of damaged astrocytes in vitro. We prepared damaged model of astrocytes to simulate the responsive hyperplasia of damaged astrocytes in vivo by culturing astrocytes in vitro; After the first day of Ad-HGF transfection, astrocytes were scratched, then after the first, the third, and the fifth day of scratch, we detect the expression amount of astrocytes specific glial fibrillary acidic protein (GFAP) and the ratio of S-phase cells with flow cytometry, both of which can reflect the proliferation status of damaged astrocytes; After HGF was added in scratched astrocytes, the activity of SPK and MAPK (P42/44) were detected by autoradiography and immunoblotting test; After adding different concentrations of HGF protein in astrocytes cultured in different serum concentrations and adding diverse concentrations of HGF protein, SPK and SPK inhibitor DMS in scratched astrocytes, we detect cell proliferation with 3H-TDR incorporation. The first day after Ad-HGF transfected astrocytes were scratched, the amount of GFAP secreted by astrocytes were decreased significantly (P < 0.05), and the cells in S phase were declined obviously. HGF has bidirectional regulation on SPK of scratched astrocytes: increases the SPK activity when HGF in low dose, while inhibits when in high dose. In addition, DMS can block the signal passage; HGF had no effects on MAPK (P42/44) of damaged astrocytes cells. In conclusion, after the transfection of Ad-HGF, it can inhibit the responsive hyperplasia of damaged astrocytes by the means of blocking SPK passage.     

Peripheral Nerve Lesion Induces an Up-regulation of Spy1 in Rat Spinal Cord

Spy1, as a member of the Speedy/RINGO family and a novel activator of cyclin-dependent kinases, was shown to promote cell cycle progression and cell survival in response to DNA damage. While its expression and roles in nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Spy1 expression in lumbar spinal cord. Temporally, Spy1 expression was increased shortly after sciatic nerve crush and peaked at day 2. Spatially, Spy1 was widely expressed in the lumbar spinal cord including neurons and glial cells. While after injury, Spy1 expression was increased predominantly in astrocytes and microglia, which were largely proliferated. Moreover, there was a concomitant up-regulation of CDK2 activity and down-regulation of p27. Collectively, we hypothesized peripheral nerve injury induced an up-regulation of Spy1 in lumbar spinal cord, which was associated with glial proliferation. Ye Huang and Yonghua Liu contributed equally to this work.     

Glial cell responses, complement and apolipoprotein J expression following axon injury in the neonatal rat

Immature motoneurons are highly susceptible to degeneration following axon injury. The response of perineuronal glia to axon injury may significantly influence neuronal survival and axon regeneration. We have examined the central reactions to neonatal facial nerve transection with emphasis on the expression of complement component C3 (C3) and the multifunctional apolipoprotein J (ApoJ). Axotomy was performed on one-day-old rats. Animals were perfused from eight hours to two weeks after the lesion. The astroglial marker, glial fibrillary acidic protein (GFAP) was increased from one day and the microglial marker OX-42 from two days after injury. ApoJ immunoreactivity was increased in axotomized neuronal perikarya and astroglial cells from one day postaxotomy, but no C3 immunoreactive profiles were found at any postoperative survival time. Cell proliferation as judged by bromodeoxyuridine labeling and immunoreactivity for the cyclin Ki-67 antigen (antibody MIB5) occurred only at two days after injury. Double immunostaining revealed that the vast majority of proliferating cells were microglia, although occasional cells double labeled astrocytes were found as well. Our results indicate that the non-neuronal response in neonatal animals differ from that of adult ones as follows: 1) microglia transform rapidly into phagocytes in parallel with the degeneration of axotomized neurons, 2) despite the presence of neuronal degeneration, no expression of C3 was found, and the upregulation of the expression of the complement C3 receptor (CR3) is delayed, 3) ApoJ is strongly upregulated in perineuronal astrocytes as well as in the axotomized motoneurons. The marked upregulation of ApoJ in both instances suggests a general role of this protein in the neuronal response to axotomy.     

p75NTR Promotes Astrocyte Proliferation in Response to Cortical Stab Wound

Astrogliosis after brain trauma can have a significant impact on functional recovery. However, little is known about the mechanisms underlying astrocyte proliferation and subsequent astrogliosis. In this study, we established a cortical stab wound injury mouse model and observed dramatic astrocyte activation and nerve growth factor receptor (p75NTR) upregulation near the lesion. We also found profound alterations in the cell cycle of astrocytes near the lesion, with a switch from a mitotically quiescent (G0) phase to the G2/M and S phases. However, no changes in the level of astrocyte apoptosis were observed. Cell cycle progression to the G2/M and S phases and CDK2 protein levels in response to cortical stab wound was inhibited after p75NTR knockdown in mouse astrocytes. Conversely, p75NTR overexpression in mouse astrocytes was sufficient in promoting cell cycle progression. In conclusion, our results suggested that p75NTR upregulation in astrocytes after brain injury induces cell cycle entry by promoting CDK2 expression and promoting astrocyte proliferation. Our findings provided a better understanding of astrocytic responses after cortical stab wound injury in mice.

     

Proliferation of glial cells induced by lithium in the neural lobe of the rat pituitary is enhanced by dehydration

Rats dehydrated by 6 days of water deprivation had a low level of mitotic activity in the astrocytes ('pituicytes') of the neural lobe of the pituitary. Mitotic activity in the pituicytes was greatly increased when isotonic lithium was administered in the last 3 days of water deprivation. Rehydration on the last day of the experiment produced a further increase in mitoses. Isotonic solutions of sodium, potassium or rubidium chloride did not increase mitoses. This model of cell proliferation is of interest because the mitotic activity is related to a physiological attempt to maintain homeostasis rather than a response to injury or the development of neoplasia.     

Increased expression of nitric oxide synthase interacting protein (NOSIP) following traumatic spinal cord injury in rats

Previous studies indicated that nitric oxide (NO) is involved in secondary damage of spinal cord injury (SCI), which worsens the primary physical injury to the central nervous systems. Recently, nitric oxide synthase interacting protein (NOSIP) has been identified to interact with neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase by inhibiting the NO production. However, its expression and function after a central nervous system injury remains unclear. In this study, we examined the expression and cellular localization of NOSIP in the spinal cord of an adult rat. Western blot analysis indicated that NOSIP protein levels increased at day1 post-injury and peaked at day 14. Double immunofluorescence staining showed that NOSIP was primarily expressed in neurons and glial cells in the intact spinal cord. Interestingly, this study also showed that the expression of NOSIP significantly increased in astrocytes after injury. Furthermore, injury-induced expression of NOSIP was co-expressed with proliferating cell nuclear antigen (PCNA) positive astrocytes after injury. We also showed the NOSIP was co-localized with nNOS in gray matter and white matter after SCI. All these data taken together suggested that NOSIP may play an important roles in astrogliogenesis after a spinal cord injury.