Melanomversorgung während eines Jahres Pandemie in Berlin: abnehmende Terminstornierungen trotz zunehmender Besorgnis über COVID-19.

HINTERGRUND UND ZIELE: Die COVID-19-Pandemie stellt für Krebspatienten eine große Herausforderung dar. Unser Ziel war es, ihren Einfluss auf die Behandlung und auf Arzttermine von Melanompatienten nach einem Jahr Pandemie zu untersuchen. PATIENTEN UND METHODIK: Melanompatienten, die im Vivantes Hauttumorzentrum in Berlin behandelt wurden, beantworteten eine postalische Umfrage zu Pandemie-bedingten Änderungen ihrer Melanomversorgung. Einflussfaktoren auf Terminänderungen wurden mit deskriptiven Analysen und multivariater logistischer Regression untersucht. Daten nach einem Jahr Pandemie wurden mit Daten nach der ersten Welle verglichen. ERGEBNISSE: Von den 366 Teilnehmern (57,7 % Männer; Durchschnittsalter 69,2 Jahre, Rücklaufquote: 36,1 %) berichteten 38 (10,1 %) über verschobene oder verpasste Arzttermine, meist auf eigenen Wunsch (71,1 %) aus Angst vor COVID-19 (52,6 %). Eine aktuelle Therapie war mit einem geringeren Risiko, Termine zu verpassen, assoziiert (Odds Ratio [OR]: 0,194, p = 0,002), höheres Alter (OR: 1,037, p = 0,039), längere Krankheitsdauer (OR: 1,007, p = 0,028) und ein höherer Schulabschluss (OR: 2,263, p = 0,043) mit höherer Wahrscheinlichkeit. Von den 177 Patienten, die aktuell eine Therapie erhielten, erfuhren nur 1,7 % Pandemie-bedingte Behandlungsänderungen. Die Besorgnis über COVID-19 war nach einem Jahr Pandemie signifikant größer als nach der ersten Welle, die Zahl der verpassten Arzttermine jedoch niedriger. SCHLUSSFOLGERUNGEN: Pandemie-bedingte Änderungen waren in unserer Kohorte selten und nahmen trotz zunehmender Besorgnis mit der Zeit ab.

Melanoma care during one year pandemic in Berlin: decreasing appointment cancellations despite increasing COVID-19 concern.

BACKGROUND AND OBJECTIVES: The COVID-19 pandemic poses a great challenge for cancer patients. Our aim was to assess its influence on treatment and appointments of melanoma patients after one year of pandemic. METHODS: Melanoma patients treated in the Vivantes Skin Cancer Centre in Berlin, Germany completed a postal survey on pandemic-related alterations in melanoma care. Impact factors on changes of appointments were examined with descriptive analyses and multivariate logistic regression. Data after one year of pandemic were compared to those after its first wave. RESULTS: Among 366 participants (57.7 % males; mean age 69.2 years, response rate: 36.1 %), 38 (10.1 %) reported postponed or missed appointments, mostly on their own demand (71.1 %) due to fear of COVID-19 (52.6 %). Current treatment was associated with a lower risk of changing appointments (Odds Ratio [OR]: 0.194, p = 0.002), higher age (OR: 1.037, p = 0.039), longer disease duration (OR: 1.007, p = 0.028), and higher school degree (OR: 2.263, p = 0.043) with higher probability. Among 177 patients currently receiving therapy, only 1.7 % experienced pandemic-related treatment alterations. Concern about COVID-19 was significantly higher after one year of pandemic than after its first wave, but the number of missed appointments was lower. CONCLUSIONS: Pandemic-related changes were rare in our cohort and decreased over time despite increasing concern.

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.

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.

Melatonin receptor 1-dependent gene expression in the mouse pars tuberalis as revealed by cDNA microarray analysis and in situ hybridization.

Melatonin is an important rhythmic endocrine signal within the circadian system of mammals. The hypophyseal pars tuberalis (PT) is a major target for melatonin and shows a high density of melatonin type 1 receptors (MT1). Melatonin affects expression of clock genes in PT cells which encode for transcriptional regulators of rhythmic gene expression. In this study, microarray analysis was performed to screen for genes coding for transcriptional regulators under the control of MT1 receptors in the mouse PT. Gene expression levels were compared between melatonin-proficient mice deficient for MT1 (MT1-/-) and the corresponding wild-type (WT) during mid-subjective day (CT06, low endogenous melatonin levels) and mid-subjective night (CT18, high endogenous melatonin levels). In situ hybridization was used to validate the data obtained by microarray analysis to analyze the acute effect of daytime melatonin application on gene expression in the wild-type PT. In the wild-type PT, expression of Tim was higher during day as compared to night. These day/night differences in gene expression were not observed in the PT of MT1-/- mice, demonstrating the impact of MT1-mediated signal transduction pathway. Day-time application of melatonin acutely affected Tim and Cry1 expression but not Neurod1 and Npas4 expression. We conclude that melatonin regulates expression of genes coding for transcriptional regulators in the PT through MT1 receptors by either acute or long-term mechanisms.

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.

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.

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.

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.

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.

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.

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.

Induction of brain-derived neurotrophic factor in plaque-associated glial cells of aged APP23 transgenic mice.

Brain-derived neurotrophic factor (BDNF) is a versatile neurotrophic factor that has been implicated in cell survival, cell differentiation, axonal growth, and activity-dependent synaptic plasticity. Changes in BDNF expression have also been reported during the course of several neurological disorders, including Alzheimer's disease (AD). The role of BDNF in AD, however, has remained elusive. To learn more about this neurotrophic factor, we investigated BDNF expression in brain of amyloid precursor protein overexpressing mice (APP23 transgenic mice). In situ hybridization revealed BDNF mRNA signals associated with amyloid plaques. Laser microdissection in combination with quantitative RT-PCR demonstrated a sixfold increase of BDNF mRNA in the immediate plaque vicinity, a threefold increase in a tissue ring surrounding the plaque, and control levels in interplaque areas comparable with those measured in age-matched nontransgenic mice. Double immunofluorescence localized BDNF to microglial cells and astrocytes surrounding the plaque. Cortical BDNF protein levels were quantified by ELISA demonstrating a >10-fold increase compared with age-matched controls. This upregulation of BDNF protein significantly correlated with the beta-amyloid load in the transgenic animals. Taken together, our data demonstrate a plaque-associated upregulation of BDNF in APP23 transgenic mice and implicate this neurotrophin in the regulation of inflammatory and axonal growth processes in the plaque vicinity.

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.

Laminar organization of the mouse dentate gyrus: insights from BETA2/Neuro D mutant mice.

The dentate gyrus of rodents is characterized by a highly laminar organization: above a compact granule cell layer, commissural/associational (C/A) fibers terminate on proximal granule cell dendrites and entorhinal fibers terminate on distal granule cell dendrites in a nonoverlapping manner. To gain insights into mechanisms that underlie the formation of this laminar structure, we studied mice deficient for BETA2/NeuroD, a basic helix-loop-helix transcription factor essential for granule cell differentiation. Anterograde tracing was used to label C/A and entorhinal fibers and combined with confocal double immunofluorescence for calbindin, calretinin, parvalbumin, and reelin to visualize putative target cells. The dentate gyrus of mutant mice contained only few granule cells, which formed a cap-like structure adjacent to area CA3. Despite the severe hypoplasia of the dentate gyrus, the remaining BETA2/NeuroD-deficient granule cells expressed mature markers, extended dendrites into the molecular layer, and extended mossy fibers into area CA3. Entorhinal and C/A fibers terminated in a nonoverlapping manner in the dendritic field overlying the rudiment. Entorhinal fibers terminated in the outermost portion of the dentate gyrus where they surrounded reelin-positive Cajal-Retzius cells, and C/A fibers terminated above and within the dentate rudiment. The laminar termination of C/A fibers was closest to normal in zones of the rudiment in which granule cells were densely packed. These data indicate that granule cells are able to differentiate in the absence of BETA2/NeuroD and suggest that the signals underlying the laminar anatomy of the dentate gyrus are present in the absence of most target cells.

Quantification of layer-specific gene expression in the hippocampus: effective use of laser microdissection in combination with quantitative RT-PCR.

Laser microdissection in combination with quantitative RT-PCR is now widely appreciated as an excellent tool for quantifying mRNA levels in defined cell populations. It may be particularly useful in the hippocampal formation, where principal cells form distinct and readily identifiable cell layers. Here we are presenting an optimized protocol for labeling hippocampal principal cells on foil-mounted sections for microdissection with the Leica AS LMD system and discuss potential further applications and pitfalls. Employing this optimized method, we studied changes in brain-derived neurotrophic factor (BDNF) mRNA expression in granule cells of the mouse dentate gyrus following unilateral entorhinal cortex lesion. In this lesioning paradigm, changes in BDNF mRNA expression have previously been reported in the rat. Using laser microdissection, the granule cell layers ipsi- and contralateral to the lesion were collected and changes in BDNF levels were quantified using quantitative RT-PCR. BDNF mRNA levels were five-fold higher on the ipsilateral side compared to levels found on the contralateral side or in controls. The development of this optimized method for laser microdissection and subsequent quantitative RT-PCR allows layer-specific quantification of gene expression levels in the hippocampus and may be similarly employed in other brain areas or tissues with a laminar arrangement or high density of cells.

Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression.

Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes (n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.