Clonal expansion of CD4+CD8+ T cells in an adult patient with Mycoplasma pneumoniae-associated Erythema multiforme majus.

BACKGROUND: Erythema multiforme (EM) is an acute, immune-mediated mucocutaneous disease, most often preceded by herpes simplex virus (HSV) infection or reactivation. Mycoplasma pneumoniae (Mp) is considered the second major trigger of EM and is often associated with an atypical and more severe presentation of disease, characterized by prominent mucosal involvement. However, contrary to HSV-associated Erythema multiforme (HAEM), immunological mechanisms of Mp-associated EM remain unclear. CASE PRESENTATION: We present the case of a 50-year-old male patient presenting with community-acquired pneumonia (CAP) and erythema multiforme majus (EMM). Acute Mp infection was diagnosed by seroconversion, with no evidence of HSV infection as a cause of EMM. We performed immune phenotyping of blister fluid (BF) and peripheral blood (PB) T cells and detected a clonally expanded TCRVß2+ T cell population that was double positive for CD4 and CD8, and expressed the cytotoxic markers granulysin and perforin. This CD4+CD8+ population comprised up to 50.7% of BF T cells and 24.9% of PB T cells. Two years prior to the onset of disease, the frequency of PB CD4+CD8+T cells had been within normal range and it gradually returned to baseline levels with the resolution of symptoms, suggesting an involvement of this population in EMM disease pathophysiology. CONCLUSIONS: This report is the first to provide a phenotypic description of lesional T cells in Mp-associated EMM. Characterizing the local immune response might help to address pathophysiological questions and warrants further systematic research.

Evaluation of optical coherence tomography as a non-invasive diagnostic tool in cutaneous wound healing.

BACKGROUND: The monitoring of wound-healing processes is indispensable for the therapeutic effectiveness and improved care of chronic wounds. Histological sections provide the best morphological assessment of wound recovery, but cause further tissue destruction and increase the risk of infection. Therefore, it is reasonable to apply a diagnostic tool that allows a non-invasive and reliable observation of morphological changes in wound healing. METHODS: Optical coherence tomography (OCT) is an imaging technique for in vivo evaluation of skin diseases with a resolution close to histopathology. The aim of this study was to investigate whether OCT is suited to display the phases of wound healing. For this purpose, six patients with chronic wounds were objectively characterized by OCT during a period of 2 weeks. RESULTS: Comparable results between histological findings and OCT were achieved. OCT allowed the detection of partial loss of the epidermis, vasoconstriction, vasodilatation and epithelialization. CONCLUSION: Consequently, OCT could be a potential non-invasive diagnostic tool for the characterization and monitoring of cutaneous wound-healing processes over time.

Celecoxib desensitization: continued temozolomide/celecoxib chemotherapy after a celecoxib-induced hypersensitivity reaction.

Cox-2 inhibitors have been identified as promising candidates for cancer therapy. Several studies have recently proposed the use of celecoxib in long-term low-intensity chemotherapy protocols for recurrent tumors. However, drug-induced hypersensitivity reactions may force discontinuation of the medication and, thus, significantly complicate successful care. Here, we report on celecoxib desensitization after a celecoxib-induced skin reaction, thereby allowing the continuation of temozolomide/celecoxib chemotherapy in a young patient with recurrent astrocytoma.

Diagnosis of drug-induced skin reactions: a future role for computer-aided systems?

PURPOSE OF REVIEW: The diagnosis of drug-induced hypersensitivity reactions is a common clinical challenge and the skin is considered a signal organ for a large percentage of these reactions. In order to assure clinical improvement of the patient, nondermatologists in primary or intensive care settings attending to patients receiving several drugs are often confronted with the need for an immediate decision which drug to stop and which drug to continue. As reliable allergological tests are missing for most drugs and are not available for immediate interventions, computer-aided diagnostic systems might be of assistance. RECENT FINDINGS: This review highlights the evolution of automated diagnostic aides in dermatology, emphasizes prerequisites for the development of appropriate computational algorithms and discusses the specific requirements and chances for the development of computer-aided diagnostic systems as a supportive approach in the diagnosis of culprit drugs for putative drug-induced hypersensitivity reactions. SUMMARY: As complex computational algorithms have to be created integrating both image and data analysis, the development of a computer-aided system for supporting nondermatologists in the diagnosis of drug-related hypersensitivity reactions is an ambitious task but might represent an achievable goal for the medium-term future.

Differential regulation of toll-like receptor mRNAs in amyloid plaque-associated brain tissue of aged APP23 transgenic mice.

Alzheimer's disease (AD) is characterized by the pathological deposition of amyloid-beta protein in the aged brain. Inefficient clearance of amyloid-beta from brain tissue is believed to play a major role in the pathogenesis of these deposits. Since amyloid-beta clearance likely involves activation of microglial cells via toll-like receptors and since these receptors and their signaling pathways are regarded as potential therapeutic targets, we have studied the expression of toll-like receptor (tlr) mRNAs in an animal model of AD (APP23 transgenic mice). Laser microdissection was used to harvest plaques, tissue surrounding plaques and plaque-free tissue from cortex of aged APP23 transgenic mice and age-matched controls. Real-time RT-PCR was employed to quantify expression levels of different tlr mRNAs in these tissues. This revealed a strong upregulation of tlr2, tlr4, tlr5, tlr7 and tlr9 mRNAs in plaque material compared to plaque-free tissue. In contrast, tlr3 was not significantly upregulated. Plaque-free tissue did not show an increased expression of any tlr mRNAs compared to age-matched control mice. Double-immunofluorescence for TLR2 and the microglial marker Iba1 was used to demonstrate localization of TLR2 on plaque-associated microglia. Taken together, these data show a strong upregulation of mRNAs encoding surface TLRs in plaque-associated brain tissue of aged APP23 transgenic mice. Since TLR-upregulation is restricted to plaques, modifying TLR-signaling may be a promising therapeutic strategy for plaque removal.

Intrastromal keratotomy with femtosecond laser avoids profibrotic TGF-beta1 induction.

PURPOSE: To examine expression of the profibrotic cytokine TGF-beta1 after selective intrastromal corneal injury with the use of a femtosecond laser. METHODS: Rabbits underwent monocular intrastromal keratotomy at a preoperatively determined corneal depth of 160 to 200 mum with the use of a femtosecond laser. Femtosecond laser-induced TGF-beta1 expression was compared in nonoperated control eyes and eyes treated with photorefractive keratectomy (PRK). Follow-up examinations were performed 1, 3, 7, and 28 days after surgery. TGF-beta1 protein was identified by immunofluorescence labeling. With the use of laser-capture microdissection, epithelial, stromal, and endothelial cell layers were collected, and changes in TGF-beta1 mRNA expression were quantified with quantitative RT-PCR. RESULTS: TGF-beta1 mRNA and protein expression did not significantly increase after intrastromal femtosecond laser keratotomy. In contrast, TGF-beta1 was induced in corneal epithelial and stromal cells after PRK and showed up to 23-fold higher TGF-beta1 mRNA levels compared with control corneas. The increase of TGF-beta1 mRNA levels after PRK was accompanied by increased TGF-beta1 protein production. CONCLUSIONS: Isolated stromal injury with a femtosecond laser does not result in induction of the profibrotic cytokine TGF-beta1. Because TGF-beta1 has been implicated in a fibrotic response of the corneal stroma to injury, absence of TGF-beta1 induction argues for a favorable wound-healing response. These findings support highly selective intrastromal procedures in refractive surgery.

TREM2 is upregulated in amyloid plaque-associated microglia in aged APP23 transgenic mice.

Alzheimer's disease (AD) is characterized by extracellular deposits of amyloid-beta protein which attract dense clusters of microglial cells. Here, we analyzed amyloid plaque-associated areas in aged APP23 transgenic mice, an animal model of AD, by combining laser microdissection with microarray analysis and quantitative RT-PCR (qPCR). By comparing gene expression profiles, we found that 538 genes (1.3% of a total of 41,234 analyzed genes) were differentially expressed in plaque-associated versus plaque-free tissue of aged APP23 transgenic mice. One of these genes is the microglia-associated triggering receptor expressed on myeloid cells (TREM2) which enhances phagocytosis, but abrogates cytokine production as well as TLR and Fc receptor-mediated induction of TNF secretion. Western Blot analysis demonstrated an upregulation of TREM2 protein in APP23 transgenic compared with nontransgenic mice. Confocal imaging studies, furthermore, confirmed colocalization of TREM2 protein with microglia. Thus, when TREM2 is induced on microglia in plaque-loaded brain areas the respective signaling may prevent inflammation-induced bystander damage of neurons. At the same time, TREM2 signaling may also account for the failure to sufficiently eliminate extracellular amyloid with the help of a systemic immune response.

Differential regulation of chondroitin sulfate proteoglycan mRNAs in the denervated rat fascia dentata after unilateral entorhinal cortex lesion.

Following brain trauma, chondroitin sulphate proteoglycans (CSPGs) are enriched at injury sites and in denervated areas. At injury sites, CSPGs are regarded as inhibitors of axonal regeneration because of their growth inhibitory properties. In areas of denervation their role is less clear, since they are enriched in zones of sprouting, i.e. zones of axonal growth. To identify CSPGs expressed in a denervated brain area and to quantify changes in their mRNA expression, neurocan, brevican, NG2, phosphacan and aggrecan mRNA were analyzed in the rat fascia dentata following entorhinal denervation. Laser microdissection was combined with quantitative RT-PCR to measure mRNA changes specifically within the denervated portion of the molecular layer (1h, 6h, 10h, 12h, 1d, 2d, 3d, 4d, 7d and 14d post-lesion). Changes in glial fibrillary protein mRNA were measured at the same time points and used as lesion control. This approach revealed a differential regulation of CSPG mRNAs in the denervated zone: neurocan, brevican and NG2 mRNA were upregulated with a maximum around 2 days post-lesion. In contrast, aggrecan mRNA levels reached a maximum 7 days post-lesion and phosphacan mRNA levels were not significantly altered. Taken together, our data reveal a temporal pattern in CSPG mRNA expression in the denervated fascia dentata. This suggests specific biological functions for CSPGs during the denervation-induced reorganization process: whereas the early increase in CSPGs in the denervated zone could influence the pattern of sprouting, the late increase of aggrecan mRNA suggests a different role during the late phase of reorganization.

3D-reconstruction and functional properties of GFP-positive and GFP-negative granule cells in the fascia dentata of the Thy1-GFP mouse.

Granule cells of the mouse fascia dentata are widely used in studies on neuronal development and plasticity. In contrast to the rat, however, high-resolution morphometric data on these cells are scarce. Thus, we have analyzed granule cells in the fascia dentata of the adult Thy1-GFP mouse (C57BL/6 background). In this mouse line, single neurons in the granule cell layer are GFP-labeled, making them amenable to high-resolution 3D-reconstruction. First, calbindin or parvalbumin-immunofluorescence was used to identify GFP-positive cells as granule cells. Second, the dorsal-ventral distribution of GFP-positive granule cells was studied: In the dorsal part of the fascia dentata 11% and in the ventral part 15% of all granule cells were GFP-positive. Third, GFP-positive and GFP-negative granule cells were compared using intracellular dye-filling (fixed slice technique) and patch-clamp recordings; no differences were observed between the cells. Finally, GFP-positive granule cells (dorsal and ventral fascia dentata) were imaged at high resolution with a confocal microscope, 3D-reconstructed in their entirety and analyzed for soma size, total dendritic length, number of segments, total number of spines and spine density. Sholl analysis revealed that dendritic complexity of granule cells is maximal 150-200 mum from the soma. Granule cells located in the ventral part of the hippocampus revealed a greater degree of dendritic complexity compared to cells in the dorsal part. Taken together, this study provides morphometric data on granule cells of mice bred on a C57BL/6 background and establishes the Thy1-GFP mouse as a tool to study granule cell neurobiology.

Corneal femtosecond laser keratotomy results in isolated stromal injury and favorable wound-healing response.

PURPOSE: To examine the corneal repair response after intrastromal femtosecond (fs) laser keratotomy. METHODS: Twelve rabbits underwent monocular intrastromal keratotomy performed with an fs laser at a preoperatively determined corneal depth of 160 to 200 microm. The fs laser-induced corneal repair response was compared with that of nonoperated control eyes and eyes treated with photorefractive keratectomy (PRK). Follow-up examinations were performed 1, 3, 7, and 28 days after surgery. Corneas were evaluated using slit lamp, in vivo confocal microscopy, and light microscopy. The extracellular matrix components fibronectin and tenascin were located using immunofluorescence staining. Anti-Thy-1 and anti-alpha-SMA antibodies and phalloidin were used to identify repair fibroblasts. Cell proliferation and nuclear DNA fragmentation were detected using an anti-Ki-67 antibody and the TUNEL assay, respectively. RESULTS: Intrastromal fs keratotomy resulted in a hypocellular stromal scar discernible as a narrow band of increased reflectivity on slit lamp examination. Deposition of fibronectin and tenascin as well as death and subsequent proliferation of keratocytes were observed. No differentiation of keratocytes into Thy-1- or alpha-SMA-positive fibroblasts could be detected. In contrast, after PRK, which causes epithelial and stromal wounding, all markers for repair fibroblasts were found in subepithelial stromal layers. On slit lamp examination, a fibrotic scar and a corneal haze were revealed. CONCLUSIONS: Isolated stromal injury using an fs laser avoids epithelial injury and is associated with a favorable wound-healing response preserving corneal transparency. Thus, fs laser keratotomy is a highly selective laser treatment that can be useful for the treatment of refractive errors.

A role for synaptopodin and the spine apparatus in hippocampal synaptic plasticity.

Spines are considered sites of synaptic plasticity in the brain and are capable of remodeling their shape and size. A molecule thathas been implicated in spine plasticity is the actin-associated protein synaptopodin. This article will review a series of studies aimed at elucidating the role of synaptopodin in the rodent brain. First, the developmental expression of synaptopodin mRNA and protein were studied; secondly, the subcellular localization of synaptopodin in hippocampal principal neurons was analyzed using confocal microscopy as well as electron microscopy and immunogold labelling; and, finally, the functional role of synaptopodin was investigated using a synaptopodin-deficient mouse. The results of these studies are: (1) synaptopodin expression byhippocampal principal neurons develops during the first postnatal weeks and increases in parallel with the maturation of spines in the hippocampus. (2) Synaptopodin is sorted to the spine compartment, where it is tightly associated with the spine apparatus, an enigmatic organelle believed to be involved in calcium storage or local protein synthesis. (3) Synaptopodin-deficient mice generated by gene targeting are viable but lack the spine apparatus organelle. These mice show deficitsin synaptic plasticity as well as impaired learning and memory. Taken together, these data implicate synaptopodin and the spine apparatus in the regulation of synaptic plasticity in the hippocampus. Future studies will be aimed at finding the molecular link between synaptopodin, the spine apparatus organelle, and synaptic plasticity.

Vessel ultrastructure in APP23 transgenic mice after passive anti-Abeta immunotherapy and subsequent intracerebral hemorrhage.

Passive immunization of amyloid precursor protein (APP) transgenic mice with anti-amyloid beta (Abeta) antibodies was shown to reduce Abeta-deposition in brain and to improve cognition. However, immunotherapy may also be accompanied by a significant increase in the frequency of intracerebral hemorrhages. Because hemorrhages are associated with amyloid-laden vessels, this raises the question whether high concentrations of anti-Abeta antibodies may directly or indirectly lead to a structural destabilization of the vessel wall. To address this point, transmission electron microscopy was performed and the ultrastructure of bleeding and non-bleeding vessels in immunized and non-immunized APP23 transgenic animals was analyzed. To localize bleeding vessels, hemosiderin-positive macrophages were visualized by pre-embedding Perl's Berlin Blue histochemistry. Vessels were analyzed morphologically, anomalies evaluated and quantified. Bleeding vessels were, furthermore, reconstructed in three dimensions to analyze the spatial distribution of amyloid deposits and other pathological changes of the vessel wall. This in-depth morphological analysis revealed that bleeding vessels in immunized as well as in non-immunized APP23 mice were surrounded by a higher number of macrophages compared to non-bleeding vessels in the same animals. However, no differences in the number of macrophages or other structural parameters, such as amyloid deposition, were observed between bleeding vessels of immunized and non-immunized mice. No pathologies which may indicate impending bleeding were observed in the vascular wall of non-bleeding vessels. We conclude, that the increased hemorrhage frequency observed after passive immunization with anti-Abeta antibodies does not lead to overt structural changes in the vessel wall of APP23 transgenic mice. Minor structural alterations of the vessel wall, however, cannot be excluded due to the sample size of our study and the high complexity of the three-dimensional vessel wall ultrastructure.

Neuronal hyperactivity induces astrocytic expression of neurocan in the adult rat hippocampus.

Extracellular matrix molecules are involved in the cellular functions of proliferation, migration, morphological differentiation, and synaptic plasticity. One candidate molecule of the extracellular matrix is the chondroitin sulfate proteoglycan neurocan. To determine whether neurocan expression is regulated by neuronal activity in the adult rat brain, we studied changes in hippocampal neurocan mRNA and protein expression following electrical stimulation of the perforant pathway in urethane-anesthetized rats. After 24 h of intermittent, unilateral 20 Hz stimulation, in situ hybridization revealed increased neurocan mRNA in glial fibrillary acidic protein (GFAP)-positive astrocytes bilaterally in all hippocampal subfields. These changes were quantified in the dentate molecular layer, the termination zone of the perforant pathway, using laser microdissection in combination with quantitative reverse transcription-polymerase chain reaction (RT-PCR). Immediately after 24 h stimulation, a six-fold upregulation was detected, which returned to control levels by 3 days post-stimulation. Neurocan immunoreactivity was similarly upregulated bilaterally. Immunostaining intensity reached a maximum by 4 days and returned to control levels by 14 days. The pattern of neurocan expression in the hippocampus depended on the intensity and duration of electrical stimulation. Under conditions of less intense afferent stimulation (4-24 h of 2.0 Hz paired-pulse stimulation, interpulse interval 40 ms), increases in neurocan mRNA and immunoreactivity were restricted to the ipsilateral termination zone of the stimulated perforant pathway. This layer-specific neurocan upregulation was not affected by intraperitoneal application of the NMDA-receptor antagonist MK-801. In conclusion, our data indicate that synaptic activity regulates the astrocytic expression of neurocan in a graded manner.

Relationship of apolipoprotein E and age at onset to Parkinson disease neuropathology.

Previous studies investigating the association between apolipoprotein E (APOE) genotypes and Parkinson disease (PD) have yielded conflicting results, and only a few have addressed APOE as a possible determinant of PD pathology. Therefore, we aimed to evaluate the relationship between APOE and PD as well as APOE and PD pathology. We studied 108 pathologically verified patients with PD and 108 controls pair-matched for age and gender. Allele frequencies of APOE differed between patients with PD and controls (p = 0.02). The frequency of epsilon4 allele increased (p = 0.01), whereas that of epsilon3 allele decreased with advancing PD pathology (p = 0.002). Only age of PD onset was an independent predictor for the rate of progression of PD pathology in which late-onset patients appeared to reach end point PD pathology more rapidly than early-onset patients (p = 0.001). In conclusion, our findings suggest that APOE may express its effect on the risk of PD by modifying the occurrence of PD pathology, but age of PD onset seems to be the principal determinant of the progression rate of PD pathology.

NG2 upregulation in the denervated rat fascia dentata following unilateral entorhinal cortex lesion.

The chondroitin sulfate proteoglycan NG2 is a component of the glial scar following brain injury. Because of its growth inhibiting properties, it has been suggested to impede axonal regeneration. To study whether NG2 could also regulate axonal growth in denervated brain areas, changes in NG2 were studied in the rat fascia dentata following entorhinal deafferentation and were correlated with the post-lesional sprouting response. Laser microdissection was employed to selectively harvest the denervated molecular layer and combined with quantitative RT-PCR to measure changes in NG2 mRNA (6 h, 12 h, 2 days, 4 days, 7 days post-lesion). This revealed increases of NG2 mRNA at day 2 (2.5-fold) and day 4 (2-fold) post-lesion. Immunocytochemistry was used to detect changes in NG2 protein (1 days, 4 days, 7 days, 10 days, 14 days, 30 days, 6 months post-lesion). NG2 staining was increased in the denervated outer molecular layer at day 1 post-lesion, reached a maximum 10 days post-lesion, and returned to control levels thereafter. Electron microscopy revealed NG2 immunoprecipitate on glial surfaces and in the extracellular matrix around neuronal profiles, indicating that NG2 is secreted following denervation. Double labeling of NG2-immunopositive cells with markers for astrocytes, microglia/macrophages, and mature oligodendrocytes suggested that NG2 cells are a distinct glial subpopulation before and after entorhinal deafferentation. BrdU labeling revealed that some of the NG2-positive cells are generated post-lesion. Taken together, our data revealed a layer-specific upregulation of NG2 in the denervated fascia dentata that coincides with the sprouting response. This suggests that NG2 could regulate lesion-induced axonal growth in denervated areas of the brain.

Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice.

The significance of the peripheral immune system in Alzheimer's disease pathogenesis remains controversial. To study the CNS invasion of hematopoietic cells in the course of cerebral amyloidosis, we used a green fluorescence protein (GFP)-bone marrow chimeric amyloid precursor protein transgenic mouse model (APP23 mice). No difference in the number of GFP-positive invading cells was observed between young APP23 mice and nontransgenic control mice. In contrast, in aged, amyloid-depositing APP23 mice, a significant increase in the number of invading ameboid-like GFP-positive cells was found compared with age-matched nontransgenic control mice. Interestingly, independent of the time after transplantation, only a subpopulation of amyloid deposits was surrounded by invading cells. This suggests that not all amyloid plaques are a target for invading cells or, alternatively, all amyloid plaques attract invading cells but only for a limited time, possibly at an early stage of plaque evolution. Immunological and ultrastructural phenotyping revealed that macrophages and T-cells accounted for a significant portion of these ameboid-like invading cells. Macrophages did not show evidence of amyloid phagocytosis at the electron microscopic level, and no obvious signs for T-cell-mediated inflammation or neurodegeneration were observed. The observation that hematopoietic cells invade the brain in response to cerebral amyloidosis may hold an unrecognized therapeutic potential.

Lamina-specific distribution of Synaptopodin, an actin-associated molecule essential for the spine apparatus, in identified principal cell dendrites of the mouse hippocampus.

Synaptopodin is an actin-associated molecule found in a subset of telencephalic spines. It is an essential component of the spine apparatus, a Ca(2+)-storing organelle and has been implicated in synaptic plasticity (Deller et al. [2003] Proc Natl Acad Sci U S A 100:10494-10499). In the rodent hippocampus, Synaptopodin is distributed in a characteristic region- and lamina-specific manner. To learn more about the cellular basis underlying this distribution, the regional, laminar, and cellular localization of Synaptopodin and its mRNA were analyzed in mouse hippocampus. First, Synaptopodin puncta densities were quantified after immunofluorescent labeling using confocal microscopy. Second, the dendritic distribution of Synaptopodin-positive puncta was studied using three-dimensional confocal reconstructions of Synaptopodin-immunostained and enhanced green fluorescence protein (EGFP)-labeled principal neurons. Synaptopodin puncta located within dendrites of principal neurons were primarily found in spines (>95%). Analysis of dendritic segments located in different layers revealed lamina-specific differences in the percentage of Synaptopodin-positive spines. Densities ranged between 37% (outer molecular layer) and 14% (stratum oriens; CA1). Finally, synaptopodin mRNA expression was studied using in situ hybridization, laser microdissection, and quantitative reverse transcriptase-polymerase chain reaction. Expression levels were comparable between all regions. These data demonstrate a lamina-specific distribution of Synaptopodin within dendritic segments of identified neurons. Within dendrites, the majority of Synaptopodin-positive puncta were located in spines where they represent spine apparatuses. We conclude, that this organelle is distributed in a region- and layer-specific manner in the mouse hippocampus and suggest that differences in the activity of afferent fiber systems could determine its distribution.

Laser microdissection reveals regional and cellular differences in GFAP mRNA upregulation following brain injury, axonal denervation, and amyloid plaque deposition.

Astrocytes are one of the major cell types responding to central nervous system injury. Upregulation of the astrocytic intermediate filament molecule glial fibrillary acidic protein (GFAP) is a key event associated with this reaction. To study the response of astrocytes to different types of brain lesions, GFAP mRNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in mouse brain following injury, axonal denervation (entorhinal cortex lesion), and amyloid plaque deposition (APP23 transgenic mice). Analysis of tissue areas surrounding a lesion revealed a 21-fold increase of GFAP mRNA in tissue surrounding an injury site, a 6-fold increase in denervated tissue areas, and a 5-fold increase in plaque containing tissue. To this GFAP mRNA increase, astrocytic proliferation and migration as well as an increase of cellular GFAP mRNA expression within astrocytes could have contributed. To determine the degree of GFAP mRNA upregulation in individual astrocytes, an immunofluorescence protocol was developed to harvest astrocytes selectively by laser microdissection and preserve intact RNA. qRT-PCR analysis of GFAP mRNA in microdissected astrocytes revealed an 82-fold increase in astrocytes surrounding an injury site, a 30-fold increase in astrocytes located in a denervation zone, and an 18-fold increase in astrocytes surrounding an amyloid plaque. These data demonstrate that GFAP mRNA is strongly upregulated within individual reactive astrocytes in response to a lesion. Because astrocytic GFAP mRNA upregulation differs among the three lesioning paradigms, we conclude that the lesion type is an important determinant of postlesional astrocytic reactivity.