NF-kappaB and IkappaBalpha expression following traumatic brain injury to the immature rat brain

NF-kappaB is one of the most important modulators of stress and inflammatory gene expression in the nervous system. In the adult brain, NF-kappaB upregulation has been demonstrated in neurons and glial cells in response to experimental injury and neuropathological disorders, where it has been related to both neurodegenerative and neuroprotective activities. Accordingly, the aim of this study was to evaluate the cellular and temporal patterns of NF-kappaB activation and the expression of its endogenous inhibitor IkappaBalpha following traumatic brain injury (TBI) during the early postnatal weeks, when the brain presents elevated levels of plasticity and neuroprotection. Our results showed that cortical trauma to the 9-day-old rat brain induced a very fast upregulation of NF-kappaB, which was maximal within the first 24 hours after injury. NF-kappaB was mainly observed in neuronal cells of the degenerating cortex as well as in astrocytes located in the corpus callosum adjacent to the injury, where a pulse-like pattern of microglial NF-kappaB activation was also found. In addition, astrocytes of the corpus callosum, and microglial cells to a lower extent, also showed de novo expression of IkappaBalpha within the time of NF-kappaB activation. This study suggests an important role of NF-kappaB activation in the early mechanisms of neuronal death or survival, as well as in the development of the glial and inflammatory responses following traumatic injury to the immature rat brain.     

Expression of the inducible nitric oxide synthase in distinct cellular types after traumatic brain injury: an in situ hybridization and immunocytochemical study

The Marmarou’s acceleration traumatic brain injury (TBI) model, in situ hybridization and immunocytochemistry were utilized to study the temporal expression of the inducible form of nitric oxide synthase (iNOS) mRNA and protein in different cellular compartments of the rat brain. Four hours following TBI, expression of iNOS was observed in the endothelial cells of cerebral blood vessels, macrophages and many cortical and hippocampal neurons. In the cortex labeled neuronal and non-neuronal cells were primarily found in the superficial layers. In the hippocampus the strongest neuronal labeling was observed in the CA1 and CA3 (lateral part) regions. By 24 h post TBI endothelial cells no longer expressed iNOS mRNA, and the macrophage and neuronal iNOS expression was reduced by 30–50%. The reduction was assessed by automated quantitation of the silver grains that occupy individual cellular profiles using an image analysis system. Immunocytochemistry revealed de novo iNOS synthesis in non-neuronal cells at the different time points, thus paralleling the changes in iNOS mRNA expression. In contrast, iNOS immunoreactivity in neurons was not observed before 24 h post TBI, suggesting failure of iNOS protein translation at 4 h after trauma. The results demonstrate complex spatial and temporal patterns of iNOS expression in discrete cellular populations, indicating different times of nitric oxide synthesis (and release) following TBI. Uncoupling of iNOS mRNA and protein synthesis in neurons suggests differential synthesis of nitric oxide in these cells as compared to non-neuronal cellular populations after trauma. Received: 27 July 1999 / Revised: 4 October 1999 / Accepted: 3 November 1999     

人脑外伤后皮层基质金属蛋白酶9的表达:定量、定位和时相

目的探讨基质金属蛋白酶9（matrix metalloproteinases-9,MMP-9）在人创伤性脑损伤（traumatic brain injury,TBI）后挫伤皮层中的表达情况,包括表达位置、表达强度和表达时相。方法挫伤区皮层的标本来自于24位TBI患者,取样时间从伤后5h到5d,利用免疫组化技术测定MMP-9的表达强度。结果在人TBI后的挫伤区皮层中,MMP-9表达明显上调,表达高峰为伤后24-48h,MMP-9主要在神经元细胞中表达,神经胶质细胞中也有少量表达。结论MMP-9在人TBI后的挫伤区皮层中表达上调,提示其可能在TBI后的病理生理过程中起着重要作用。     

Postsynaptic localization of CB2 cannabinoid receptors in the rat hippocampus

The expression of CB2 cannabinoid receptors (CB2-Rs) in the brain and their neuronal function has now attracted research interest, since we and others have demonstrated the presence of CB2-Rs in neuronal and glial cells in the brain. In this study, we show the subcellular distribution of CB2-Rs in neuronal, glial, and endothelial cells in the rat hippocampus using immunohistochemical electron microscopy. Brain sections from the hippocampus were immunolabeled for CB2-R, visualized, and analyzed by electron microscopy. We found that in neurons, CB2-R immunoreactivity is present in the cell body as well as in large and medium-sized dendrites. In the soma, the CB2-R labeling is associated with the rough endoplasmic reticulum and Golgi apparatus demonstrating that CB2-Rs are synthesized by hippocampal neurons. CB2-R labeling in dendrites was observed in the cytoplasm and associated with the plasma membrane near the area of synaptic contact with axon terminals indicating a postsynaptic distribution of these receptors. In CB2-R immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. These results provide the first ultrastructural evidence that CB2-Rs are mainly postsynaptic in the rat hippocampus.     

Apoptosis and expression of p53 response proteins and cyclin D1 after cortical impact in rat brain

We measured the temporal profile and cellular identification of apoptosis in rat brain after cortical contusion injury. Double staining immunohistochemistry was also used to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd45, PCNA) and cell cycle protein, Cyclin D1, in male Wistar rats 48 h after injury. Cortical contusion was induced in male Wistar rats with a pneumatic impactor device. The animals were sacrificed at different times after trauma (1, 2, and 14 h and 1, 2, 4, 7 and 14 days; n=4 per time point). Sham-operated rats (n=4) and normal rats not subjected to any surgical procedure (n=4) were used as controls for temporal profile determination. Additional 11 rats were used for study of protein expression. Coronal brain sections were analyzed using an in situ terminal deoxynucleotdyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL), hematoxylin, and immunohistochemical double staining methods. Apoptotic cells were observed as early as 2 h after the impact. Apoptotic cell death peaked at 2 days, gradually tapering off afterward, although scattered apoptotic cells were detected at 2 weeks after the impact. The number of apoptotic cells at 2 days far exceeded their number at other times (p=0.009). Apoptotic cells were observed primarily in the cortex adjacent to the site of injury. In addition, apoptotic cells in conjunction with few injured cells were present in the ipsilateral hippocampus and localized to the granule layer of dentate gyrus. Our data indicate that DNA fragmentation is present in nearly all neurons subacutely after cortical contusion and persists for at least 2 weeks thereafter. Apoptosis is also present in neurons localized to the hilus of the dentate gyrus at a site remote from the area of injury suggesting a selective role for apoptosis in promoting secondary brain damage and dysfunction after traumatic brain injury. Using double staining, we were able to show that a great majority of apoptotic cells (>95%) were neurons and the rest were astrocytes and endothelial cells. Proteins associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd 45, PCNA) were expressed in the cytoplasm of normal cells of naive and sham rats. These proteins were translocated to the nuclei of apoptotic and injured cells at 48 h after cortical contusion. Cyclin D1 was not present in apoptotic cells. The differential expression of proteins associated with DNA damage, repair and the cell cycle protein Cyclin D1 in the contused brain suggest a potential role for these proteins in cell survival and apoptosis after cortical contusion.     

Erythropoietin requires NF-kappaB and its nuclear translocation to prevent early and late apoptotic neuronal injury during beta-amyloid toxicity

No longer considered exclusive for the function of the hematopoietic system, erythropoietin (EPO) is now considered as a viable agent to address central nervous system injury in a variety of cellular systems that involve neuronal, vascular, and inflammatory cells. Yet, it remains unclear whether the protective capacity of EPO may be effective for chronic neurodegenerative disorders such as Alzheimer's disease (AD) that involve beta-amyloid (Abeta) apoptotic injury to hippocampal neurons. We therefore investigated whether EPO could prevent both early and late apoptotic injury during Abeta exposure in primary hippocampal neurons and assessed potential cellular pathways responsible for this protection. Primary hippocampal neuronal injury was evaluated by trypan blue dye exclusion, DNA fragmentation, membrane phosphatidylserine (PS) exposure, and nuclear factor-kappaB (NF-kappaB) expression with subcellular translocation. We show that EPO, in a concentration specific manner, is able to prevent the loss of both apoptotic genomic DNA integrity and cellular membrane asymmetry during Abeta exposure. This blockade of Abeta generated neuronal apoptosis by EPO is both necessary and sufficient, since protection by EPO is completely abolished by co-treatment with an anti-EPO neutralizing antibody. Furthermore, neuroprotection by EPO is closely linked to the expression of NF-kappaB p65 by preventing the degradation of this protein by Abeta and fostering the subcellular translocation of NF-kappaB p65 from the cytoplasm to the nucleus to allow the initiation of an anti-apoptotic program. In addition, EPO intimately relies upon NF-kappaB p65 to promote neuronal survival, since gene silencing of NF-kappaB p65 by RNA interference removes the protective capacity of EPO during Abeta exposure. Our work illustrates that EPO is an effective entity at the neuronal cellular level against Abeta toxicity and requires the close modulation of the NF-kappaB p65 pathway, suggesting that either EPO or NF-kappaB may be used as future potential therapeutic strategies for the management of chronic neurodegenerative disorders, such as AD.     

共聚焦激光扫描显微镜研究大鼠脑缺血谷氨酸载体GLAST mRNA和EAAT1的表达与其细胞分布

目的通过应用共聚焦激光扫描显微镜技术(confocal laser scanning microscope, CLSM)观察脑缺血后谷氨酸载体GLAST mRNA和EAAT1蛋白表达的细胞定位,探讨CLSM技术中三维重建和三维显示在观察基因和蛋白在神经细胞上空间定位的应用.方法对脑缺血后采用原位杂交和免疫组织化学相结合的荧光双标技术标记的脑片进行双通道断层扫描以及三维数据重组.结果脑缺血后,荧光免疫组化双标显示大脑皮质缺血半暗区内的谷氨酸载体EAAT1蛋白与星形胶质细胞和神经元均有共表达;原位杂交结合免疫组化的荧光双标显示缺血周边区谷氨酸载体GLAST mRNA与星形胶质细胞和神经元有明显的共表达.结论共聚焦激光扫描显微镜观察脑缺血后谷氨酸载体GLAST mRNA和EAAT1蛋白在神经胶质细胞和神经元上的空间定位,为进一步研究脑缺血后谷氨酸载体系统作用机制提供了更为准确的形态依据.     

Expression patterns of 5‐lipoxygenase in human brain with traumatic injury and astrocytoma

5‐Lipoxygenase (5‐LOX) is a key enzyme in the metabolism of arachidonic acid to leukotrienes. The levels of leukotrienes increase after brain injury and when tumors are present. It has been reported that 5‐LOX is widely expressed in the brain and that 5‐LOX inhibition provides neuroprotection. However, there is still no information available for the expression patterns of 5‐LOX in human brain following trauma or with astrocytomas. We investigated its expression patterns by immunohistochemistry. We found that 5‐LOX is normally expressed in neurons and glial cells. In neurons, it was expressed in two patterns: in the cytosol and nucleus or only in the cytosol. In traumatic brain injury, 5‐LOX expression increased in glial cells and neutrophils. Double‐labeling immunohistochemistry showed that part of the 5‐LOX‐positive glial cells were GFAP positive. No 5‐LOX expression was found in brain microvessel endothelia, except in the regenerated endothelia of a patient 8 days following brain trauma. Furthermore, 5‐LOX expression increased and showed a granular pattern in high‐grade (grade III/IV) astrocytoma. These results indicate that 5‐LOX has multiple expression patterns, and can be induced by brain injury, which implies that 5‐LOX might have pathophysiological roles in the human brain.     

The presence of delta and mu-, but not kappa or ORL(1) receptors in bovine pinealocytes

Recent studies suggest that nuclear factor NF-kappaB may be involved in excitotoxin-induced cell apopotosis. To analyze the variation of NF-kappaB, levels of NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50 antibodies, respectively. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that ischemia-induced NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo, N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist nifedipine were given 20 min prior to 30 min of ischemia. The NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after ischemia/reperfusion 6 h. Increased phosphorylation of the NF-kappaB regulatory unit IkappaB-alpha was detected by Western blot. Decrement of IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global ischemia, which was also blocked by such three antagonists. These results illustrate that glutamate-gated ionotropic NMDA or non-NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate NF-kappaB activation during brain ischemic injury. Active NF-kappaB may attend the excitotoxin-induced cell death in turn. Our studies also suggest that IkappaB-alpha is an important regulatory unit that controls the activation of NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global ischemia.     

Early release of HMGB-1 from neurons after the onset of brain ischemia.

The nuclear protein high-mobility group box 1 (HMGB-1) promotes inflammation in sepsis, but little is known about its role in brain ischemia-induced inflammation. We report that HMGB-1 and its receptors, receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), and TLR4, were expressed in normal brain and in cultured neurons, endothelia, and glial cells. During middle cerebral artery occlusion (MCAO), in mice, HMGB-1 immunostaining rapidly disappeared from all cells within the striatal ischemic core from 1 h after onset of occlusion. High-mobility group box 1 translocation from nucleus to cytoplasm was observed within the cortical periinfarct regions 2 h after ischemic reperfusion (2 h MCAO). High-mobility group box 1 predominantly translocated to the cytoplasm or disappeared in cells that colabeled with the neuronal marker NeuN. Furthermore, RAGE was robustly expressed in the periinfarct region after MCAO. Cellular release of HMGB-1 was detected by immunoblotting of cerebrospinal fluid as early as 2 h after ischemic reperfusion (2 h MCAO). High-mobility group box 1 released from neurons, in vitro, after glutamate excitotoxicity, maintained biologic activity and induced glial expression of tumor necrosis factor alpha (TNFalpha). Anti-HMGB-1 antibody suppressed TNFalpha upregulation in astrocytes exposed to conditioned media from glutamate-treated neurons. Moreover, TNFalpha and the cytokine intercellular adhesion molecule-1 increased in cultured glia and endothelial cells, respectively, after adding recombinant HMGB-1. In conclusion, HMGB-1 is released early after ischemic injury from neurons and may contribute to the initial stages of the inflammatory response.     

颅脑损伤后p38丝裂原活化蛋白激酶在挫伤皮层中的表达及其意义

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）在颅脑损伤后挫伤皮层中的表达。方法挫伤皮层标本来自24例颅脑损伤患者，取样时间为伤后5h-5d，将患者按伤后取标本时间平均分为4组，即〈24h组、24～48h组、48-72h组和〉72h组。所有病例常规行开颅血肿清除术，应用免疫组化技术测定挫伤皮层中磷酸化p38MAPK的表达。结果在颅脑损伤后挫伤皮层中．p38MAPK的表达明显上调（P〈0．05），表达高峰为伤后24h内（P〈0．01），主要在血管内皮细胞中表达，在神经细胞及神经胶质细胞中极少表达。结论p38MAPK在人颅脑损伤后挫伤皮层中的表达上调，提示其可能在颅脑损伤后的病理生理过程中起重要作用。     

Activation of nuclear factor-kappaB in the rat brain after transient focal ischemia

Nuclear factor-kappaB (NF-kappaB) becomes activated under inflammatory conditions and triggers induction of gene expression. Here, activation of NF-kappaB was studied after transient middle cerebral artery occlusion in the rat. Expression of p65 and p50, protein subunits of NF-kappaB, was examined by Western blotting, and immunohistochemistry for p65 was carried out. Double-labelling with specific markers for astroglia and microglia was used for cell type identification. Neurons located within and surrounding the ischemic core were identified during the first 24 h post-ischemia by using an antibody against 72-kDa heat shock protein. NF-kappaB binding activity was evaluated at different times post-ischemia with electrophoretic mobility gel shift assays. The results showed constitutive expression of p65 and p50, and NF-kappaB binding activity. Basal p65 was seen in certain neurons and resting astrocytes. Constitutive NF-kappaB binding activity was attributable to one main protein complex possibly formed in neurons and astrocytes, although two minor complexes were also detected. At 1 day post-ischemia selective induction of p65 was seen in neurons located in a penumbra-like area. At this time, however, no disturbances of basal NF-kappaB binding activity were found. Western blotting showed delayed induction of p65 several days after ischemia, whereas no changes were detected for p50. From 4 days post-ischemia, a substantial increase in the amount of p65 was detected due to induction in reactive astrocytes and microglia/macrophages. This was correlated with a robust enhancement of NF-kappaB binding activity with formation of three major specific complexes binding DNA. It is proposed that the highly inducible NF-kappaB complexes resulted from induction of p65 and activation of NF-kappaB in post-ischemic reactive glia.     

Appearance of neuronal S-100β during development of the rat brain

In addition to being an astroglial protein, S-100β is localised in distinct populations of neurons in the adult rat hindbrain. We report, here, the expression of S-100β in both neurons and glia of the rat brain during development. Prenatally, S-100β immunoreactivity was confined to glial cells close to the germinal zone. After birth, S-100β positive glial cells were seen mainly in the brainstem and cerebellum, while only a few were detected in cerebral cortex and hippocampus. The number of S-100β containing glial cells increased steadily during the first 2 postnatal weeks after which the adult pattern was attained. No S-100β containing neurons were present prenatally. The first S-100β containing neurons were seen in the mesencephalic trigeminal nucleus at postnatal day 1 (P1), and in the motor trigeminal nucleus at P3. Neuronal S-100β immunoreactivity in other nuclei was mostly attained from the 10th to the 21st postnatal day. The neuronal S-100β immunoreactivity was first detected in the cell nuclei during development, then increased in the cytoplasm with ages. A nuclear staining in many immunoreactive neurons persisted until the adult. It usually took 1 to 2 weeks for neuronal S-100β to attain the adult staining pattern, i.e., heavy staining of the cytoplasm and processes, after its appearance. The forebrain never contained S-100β positive neurons. The S-100β is first expressed in glial cells, suggesting it is primarily of the glial origin. Coupled with neurotrophic effects of the protein, the time course of neuronal S-100β expression during the critical period of neuronal development implies that it may be involved in neuronal differentiation and maturation.     

人脑挫裂伤早期HMGB1的表达变化特征

目的探讨人脑挫裂伤早期HMGB1表达的变化特征。方法收集人脑挫裂伤后不同时间点挫裂伤组织的标本(正常组、6h、12h、24h、72h),4%多聚甲醛固定,冰冻切片,然后进行免疫荧光染色,统计分析各时间点HMGB1的表达情况;同时将HMGB1与神经元标志物NeuN进行共标,观察人脑挫裂伤后早期神经元损伤过程中从核内向胞浆内转移的情况。结果正常人脑组织的HMGB1表达主要集中在细胞核内,与正常组相比较,人脑挫裂伤组在伤后HMGB1阳性细胞数于12h开始下降(P0.05),并持续至72h;而HMGB1从核内转移至胞浆内的数目从6h即开始增加(P0.05),随时间增加HMGB1也随之大量转移到胞浆中,在人脑挫裂伤后24h达到高峰(P0.05)。人脑挫裂伤后早期,HMGB1主要表达在神经元中(P0.05),脑挫裂伤后神经元的数目逐渐减少(P0.05),而HMGB1从神经元核内转移至胞浆的比例在6h明显增加(P0.05),于24~72h达到高峰(P0.05)。结论人脑挫裂伤后早期神经元死亡或凋亡数目明显增加,HMGB1阳性细胞数也在损伤后减少;而HMGB1神经元从核内转移至胞浆的比例升高;HMGB1可能参与了脑挫裂伤后神经元的死亡或凋亡。     

Increase in phosphorylation of PDK1 and cell survival after acute spinal cord injury

3-Phosphoinositidedependent protein kinase-1 (PDK1), which phosphorylates and activates a group of kinases, plays important roles in cellular metabolism, growth, proliferation and survival. However, the functions of PDK1 in central nervous system (CNS) injury remain an enigma. To elucidate the expressions and possible functions of PDK1 and its phosphorylation in CNS injury and repair, we performed an acute spinal cord injury (SCI) model in adult rats and detected the expression and localization of serine-241 phosphorylated PDK1 (p-PDK1s241). Western blot and immunohistochemistry showed that serine-241 phosphorylated PDK1 (p-PDK1s241) started increasing by 6h after damage and peaked at 12h, then declined to basal levels by 3days after injury. Immunohistochemical staining also revealed subcellular localization changes of p-PDK1s241 staining between nucleus and cytoplasm after injury including neurons and glial cells. Double immunofluorescence labeling suggested that p-PDK1s241 primarily localizes in neurons and oligodendrocytes. It might also be expressed in other glial cells of spinal cord tissues within 2mm from the epicenter at 12h post-injury. Moreover, double staining indicated that p-PDK1s241 and active caspase-3 showed different cellular distributions after SCI. Together with previous reports, we hypothesize that phosphorylation of PDK1 may be associated with cell survival and suggest PDK1 as a novel target for neuroprotection and functional repair in SCI.     

Nuclear factor-kappaB p65 and upregulation of interleukin-6 in retinal ischemia/reperfusion injury in rats

We previously demonstrated that endogenous interleukin-6 (IL-6) is upregulated and may be neuroprotective after retinal ischemia. The purpose of this study is to investigate the role of nuclear factor kappa-B (NF-kappaB) in regulating IL-6 expression after ischemia. NF-kappaB p65 mRNA levels were significantly elevated between 2 and 12 h after the insult. A high number of NF-kappaB p65 positive cells were detected in the inner retina at 12 h after ischemia. Activated nuclear NF-kappaB p65 and IL-6 were colocalized in cells, which were also marked by a microglial/phagocytic cell marker (ED1) in the inner retina. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132, a proteasome inhibitor, which inhibits IkappaB degradation and hence prevents the activation and translocation of NF-kappaB into the nucleus) abolished the increase in NF-kappaB p65 mRNA levels after the insult, while there was no effect by helenalin (an inhibitor which inhibits NF-kappaB activity by alkylation of the p65 subunit, thereby blocking its binding to the target DNA). However, MG-132 and/or helenalin significantly diminished the increase in IL-6 mRNA levels after the insult. Small interfering RNAs (siRNAs, inhibit target gene expression through the sequence-specific destruction of the target messenger RNA) against NF-kappaB p65 significantly reduced the increase in NF-kappaB p65 mRNA levels as well as IL-6 mRNA levels after ischemia. The number of retinal ganglion cells (RGCs) was also significantly decreased using the inhibitors of NF-kappaB compared with those of the controls after ischemia. These findings support the hypothesis that upregulation of endogenous retinal IL-6 in retinal I/R injury in microglial/phagocytic cells is controlled predominantly by NF-kappaB p65.     

正常大鼠脑组织伽玛刀照射后Fos蛋白的表达及变化

目的:观察伽玛刀(γ-刀)照射正常大鼠脑部后,脑内神经元、神经胶质细胞和血管内皮细胞内Fos蛋白的表达及变化。方法:45只正常成年大鼠在脑部受γ-刀照射(100Gy),分别成活0.5h、1h、3h、6h、12h、1d、3d、7d、14d、30d至3个月后处死,灌流固定,冰冻切片,用抗Fos蛋白的免疫组织化学方法对切片进行观察。结果:在照射靶区和非靶区的大脑皮质等结构内的神经元、神经胶质细胞和血管内皮细胞均出现Fos阳性表达。Fos表达有二次高峰,第一次在3～24h之间,均为胞核染色;第二次在14天后,出现三种阳性细胞,第一种仅胞核染色,第二种胞浆染色,第三种胞核、胞浆均染色。结论:作者认为第一种者为正常反应细胞,第二种者为严重受损细胞,第三种者为中间状态细胞。