Paraffin-embedded Brain Research Articles

MCI-186 administered to the maternal circulation inhibits fetal brain injury resulting from total umbilical cord occlusion in the chronically instrumented fetal lamb

Objective. To evaluate the transplacental effects of MCI-186 (edaravone), a potent hydroxyl radical scavenger, administered to the maternal circulation to inhibit fetal brain injury caused by umbilical cord occlusion.Methods. Nine chronically instrumented lambs were prepared. In three cases, 10-min persistent total umbilical cord occlusion (group A) was performed. Another three cases underwent occlusion and were administered 60 mg of MCI-186 through the maternal femoral vein prior to the end of occlusion (group B). The remaining three cases underwent sham operation (group C). On day 3 after insult, fetal brains were extirpated. Paraffin-embedded brain tissue sections were stained with hematoxylin and eosin, Bodian, Kluver–Barrera, and TUNEL. Neuronal cellular damage was evaluated by two pathologists blinded to the experimental conditions.Results. Group A displayed numerous cells with eosinophilic condensation of nuclear chromatin and proliferation of microglia in the hippocampus and basal ganglia. TUNEL-positive cells were observed in the periventricular area. Group B showed microglial proliferations, but no marked changes. No pathological changes were apparent in group C.Conclusions. MCI-186 administered to the maternal circulation could inhibit fetal brain injury resulting from hypoxia-reperfusion induced by umbilical cord occlusion.

Disease-related Modifications in Tau Affect the Interaction between Fyn and Tau

Microtubule-associated protein tau is the major component of the neurofibrillary tangles of Alzheimer disease (AD) and is genetically linked to frontotemporal dementias (FTDP-17). We have recently shown that tau interacts with the SH3 domain of Fyn, an Src family non-receptor tyrosine kinase, and is tyrosine-phosphorylated by Fyn on Tyr-18. Also, tyrosine-phosphorylated tau is present in the neuropathology of AD. To determine whether alterations in the tau-Fyn interaction might correlate with disease-related factors in AD and FTDP-17, we have performed real-time surface plasmon resonance studies on a panel of 21 tau constructs with Fyn SH3. We report that the interaction between Fyn SH3 and 3R-tau was 20-fold higher than that with 4R-tau. In addition, the affinity between 4R-tau and Fyn SH3 was increased 25-45-fold by phosphorylation-mimicking mutations or by FTDP-17 mutations. In vitro kinase reactions show that tau, with lower affinity SH3 interactions, exhibited a lower level of Tyr-18 phosphorylation under our reaction conditions. Lastly, we have demonstrated that tau is phosphorylated on Tyr-18 in the tau P301L mouse model for tauopathy (JNPL3). In summary, our results suggest that disease-related phosphorylation and missense mutations of tau increase association of tau with Fyn. Because these effects are mediated through the 4R component of the tau population, these results also have implications for the FTDP-17 diseases caused by increased expression of 4R-tau. Our data support a role for the Fyn-tau interaction in neurodegeneration.

Doublecortin is preferentially expressed in invasive human brain tumors

Doublecortin (DCX) is required for neuroblastic migration during the development of the cerebral cortex. DCX is a microtubule-associated protein that plays a role in cellular motility. These facts led us to hypothesize that DCX is increased in invasive brain tumors. DCX expression was assessed in 69 paraffin-embedded brain tumors of neuroepithelial origin. In addition, mouse brain sections of the subventricular zone and dentate gyrus were used as positive controls for immunostaining, and specificity of antibody staining was demonstrated by peptide neutralization. DCX was highly expressed in both high-grade invasive tumors (glioblastoma, n=11; anaplastic astrocytoma/oligoastrocytoma, n=7; and medulloblastoma/PNET, n=6) and low-grade invasive tumors (oligodendroglioma, n=3; and astrocytoma/oligoastrocytoma, n=5). However, DCX was less intensely expressed in the circumscribed group of tumors (pilocytic astrocytoma, n=6; ependymoma/subependymoma, n=7; dysembryoplastic neuroepithelial tumor, n=4; ganglioglioma, n=2; meningioma, n=9; and schwannoma, n=9). By the Cochran-Mantel-Haenszel statistical test, the circumscribed group was significantly different from both the high-grade invasive group (P<0.0001) and the low-grade invasive group (P<0.0001). We conclude that DCX is preferentially expressed in invasive brain tumors. In addition, DCX immunostaining was stronger at the margin of the tumor than at the center. For a subset of these tumors, we also detected DCX mRNA and protein by Northern and Western blotting. DCX mRNA and protein was detected in glioma cell lines by Northern blotting, immunofluorescence microscopy and Western blotting. Collectively, the immunohistochemistry, Western blots and Northern blots conclusively demonstrate expression of DCX by human brain tumors.

259 RBM3 Expression in Neonates with Pontosubicular Neuron Necrosis

Objective: Pontosubicular neuron necrosis (PSN) represents an age-specific response to severe hypoxic-ischemic injury (HII) occurring in human neonates but not in older children or adults. Histologically, PSN is characterised by acute neuronal death in the pontine nuclei and the hippocampal subiculum bearing the hallmarks of apoptosis. The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under hypoxic conditions and independent of HIF (hypoxia inducible factor). It is well known, that proteins which are dependant on HIF, e.g. Erythropoetin play an important role in HII. This study aims to determine whether the HIF-independent activation of Rbm3 is also a significant factor in the pathogenesis of PSN. Methods: We have investigated the expression of Rbm3 in human autopsy material consisting of 12 PSN cases and 10 age-matched controls without PSN. Immunohistochemistry and double labeling for Rbm3 and the astrocyte marker glial fibrillary acid protein (GFAP), the microglia/macrophage specific marker KiM1P and the neuronal marker NeuN was performed on formalin-fixed, paraffin-embedded brain specimens.Results: In PSN cases and controls, mainly neuronal cells expressed Rbm3. The number of immunopositive cells was significantly increased (p=0.001) in PSN cases. Predominantly degenerating cells with signs of later apoptotic stages showed Rbm3 expression. In earlier stages of apoptosis immunopositivity for Rbm3 was increased compared to contols, but less prominent.Conclusion: In addition to HIF-dependant proteins, the induction of the HIF-independent protein RMB3 is observed in response to human hypoxic-ischemic injury.

Apolipoprotein D Expression in Primary Brain Tumors: Analysis by Quantitative RT-PCR in Formalin-fixed, Paraffin-embedded Tissue

Apolipoprotein D (apoD) expression has been shown to correlate both with cell cycle arrest and with prognosis in several types of malignancy, including central nervous system astrocytomas and medulloblastomas. ApoD expression was investigated by real-time quantitative RT-PCR using RNA extracted from 68 formalin-fixed, paraffin-embedded brain specimens. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Quantitation was achieved on all specimens. Sixteen poorly infiltrating WHO grade I glial neoplasms (i.e., pilocytic astrocytomas and gangliogliomas) showed an average 20-fold higher apoD expression level compared with the 20 diffusely infiltrating glial neoplasms (i.e., glioblastoma, anaplastic astrocytoma, oligodendrogliomas; p=0.00004). A small number of exceptions (i.e., two high-expressing glioblastomas and three low-expressing gangliogliomas) were identified. Analyzed as individual tumor groups, poorly infiltrating grade I pilocytic astrocytomas and gangliogliomas differed significantly from each tumor type within the diffusely infiltrating higher-grade category (p<0.05 for each comparison) but not from each other (p>0.05). Conversely, each individual tumor type within the diffusely infiltrating category differed significantly from both pilocytic astrocytomas and gangliogliomas (p<0.05) but did not vary from other infiltrating tumors (p>0.05). Ependymomas, non-infiltrating grade II neoplasms, expressed levels of apoD similar to or lower than levels expressed by the diffusely infiltrating gliomas. Ten medulloblastomas with survival longer than 3 years averaged slightly higher apoD expression than four fatal medulloblastomas; however, this result was not statistically significant and individual exceptions were notable. In 17 of the medulloblastomas, MIB-1 proliferation rates quantitated by image cytometry did not correlate with apoD expression. In addition, apoD expression was 5-fold higher in the slowly proliferating grade I glial neoplasms compared with non-proliferating normal brain tissue (p=0.01), suggesting that apoD expression is not simply an inverse measure of proliferation. ApoD expression measured by quantitative RT-PCR may be useful in the differential diagnosis of primary brain tumors, particularly pilocytic astrocytomas and gangliogliomas.

Neurochondrin Negatively Regulates CaMKII Phosphorylation, and Nervous System-specific Gene Disruption Results in Epileptic Seizure

Neurochondrin is a novel cytoplasmic protein and possibly involved in neurite outgrowth, chondrocyte differentiation, and bone metabolism. Our previous trial in disclosing its role by the loss of function in mice failed because of the lethality in utero. In this study, we eliminated the neurochondrin gene expression preferentially in the nervous system by the conditional knockout strategy. Our results showed that neurochondrin is a negative regulator of Ca(2+)/calmodulin-dependent protein kinase II phosphorylation and essential for the spatial learning process but not for the differentiation or neurite outgrowth of the neuron. In addition, the nervous system-specific homozygous gene disruption resulted in epileptic seizure.

A Novel Marker for Purkinje Cells, KIAA0864 Protein. An Analysis Based on a Monoclonal Antibody HFB-16 in Developing Human Cerebellum

In the search for immunohistochemical markers of the developing human brain, a monoclonal antibody, HFB-16, was raised against homogenates from the cerebrum of a 15-gestational-week-old (GW) human fetus and screened on paraffin-embedded human embryonic brain specimens. This antibody was particularly useful as a marker for Purkinje cells in the developing human cerebellum. Positive immunoreactivities with HFB-16 first appeared in the Purkinje cell layer at 17 GW. From 20 to 24 GW, positive immunoreactivities were found above the lamina dissecans. After 25 GW, dendrites of Purkinje cells were found with the HFB-16 antibody, and the nerve fibers of the Purkinje cells became positive after 35 GW. Neurons in the dentate nucleus and external and internal granular layers reacted negatively to this antibody. After 1 year, when the external granular layer faded out, the dendrites of the Purkinje cells reached the pial surface of the cerebellum, and nerve fibers began to develop in the white matter. This antibody was also useful for characterization of components in heterotopic neurons found in various anomaly syndromes such as trisomy 13. Expressional cloning indicated the antigen against HFB-16 to be human KIAA0864 protein, which is supposed to be an alternative splicing product of p116Rip, whose function has not yet been elucidated. The antigenicity of the KIAA0864 protein was confirmed using human cDNA of the KIAA0864 protein, a protein expression vector, and an HFB-16 antibody.

Immunolocalization of MAP-2 in Routinely Formalin-Fixed, Paraffin-Embedded Guinea Pig Brain Sections Using Microwave Irradiation: A Comparison of Different Combinations of Antibody Clones and Antigen Retrieval Buffer Solutions

The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.

Neuroprotective Anti-Apoptosis Effect of Estrogens in Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of death and functional disability in western countries, affecting mostly young patients. Despite intense and sustained efforts deployed for the development of new therapeutic strategies, no clinical benefit has been shown by any of the investigated compounds. Increasing attention has been drawn during the past two decades to the neuroprotective effects of estrogens, although most of the available data relate to ischemic brain injury. The purpose of the present study was to investigate the potential neuroprotective value of estrogens in TBI as a therapeutic modality. For this purpose, a contusion was created in the parietal cortex by dynamic cortical deformation in two groups of 10 Sprague-Dawley male rats. Following the injury, treated animals received conjugated estrogens for 3 days, using a subcutaneously implanted osmotic pump. Animals were then sacrificed, and TUNEL, anti-active Caspase 3, bcl-2, and bax labeling were performed in paraffin-embedded brain sections, allowing for comparative and quantitative analysis. In estrogen-treated animals, there was a marked and significant reduction of apoptosis in comparison with non-treated animals. The reduction in TUNEL and active Caspase 3 staining was similar and close to 50%. Optical analysis of histological slides prepared by bcl-2 labeling showed a significant increase in bcl-2 expression in estrogen-treated animals compared to non-treated animals. On the contrary, bax expression was not influenced by hormonal treatment, and no difference could be noticed between the two groups. These results support the potential therapeutic value of estrogens in TBI and further clarify their mode of action.

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at −80 °C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157–1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669–2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354–365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences (‘original’ sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1–6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1–5 and 10–13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1–6 substitutions, but a shift in JCV genotypes was observed in sequences with 10–13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.

Cellular and molecular tunnels surrounding the forebrain commissures of human fetuses

Glial cells and extracellular matrix (ECM) molecules surround developing fiber tracts and are implicated in axonal pathfinding. These and other molecules are produced by these strategically located glial cells and have been shown to influence axonal growth across the midline in rodents. We searched for similar cellular and molecular structures surrounding the telencephalic commissures of fetal human brains. Paraffin-embedded brain sections were immunostained for glial fibrillary acidic protein (GFAP) and vimentin (VN) to identify glial cells; for microtubule-associated protein-2 (MAP-2) and neuronal nuclear protein (NeuN) to document neurons; for neurofilament (NF) to identify axons; and for chondroitin sulfate (CS), tenascin (TN), and fibronectin (FN) to show the ECM. As in rodents, three cellular clusters surrounding the corpus callosum were identified by their expression of GFAP and VN (but not MAP-2 or NeuN) from 13 to at least 18 weeks postovulation (wpo): the glial wedge, the glia of the indusium griseum, and the midline sling. CS and TN (but not FN) were expressed pericellularly in these cell groups. The anterior commissure was surrounded by a GFAP+/VN+ glial tunnel from 12 wpo, with TN expression seen between the GFAP+ cell bodies. The fimbria showed GFAP+/VN+ cells at its lateral and medial borders from 12 wpo, with pericellular expression of CS. The fornix showed GFAP+ cells somewhat later (16 wpo). Because these structures are similar to those described for rodents, we concluded that the axon guiding mechanisms postulated for commissural formation in nonhuman mammals may also be operant in the developing human brain.

Polymerase Chain Reaction Screening for DNA Viruses in Paraffin-Embedded Brains from Dogs with Necrotizing Meningoencephalitis, Necrotizing Leukoencephalitis, and Granulomatous Meningoencephalitis

The objective of this investigation was to determine whether or not herpesvirus (herpes-), adenovirus (adeno-), or canine parvovirus DNA is present in the brains of dogs with necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE), and granulomatous meningoencephalitis (GME). Paraffin-embedded brain specimens from 12 histopathologically confirmed dogs with NME, 3 with NLE, and 7 with GME were screened for viral DNA with degenerate herpes- and adenovirus polymerase chain reaction (PCR) and a canine parvovirus-specific PCR. Positive-control specimens included genomic viral DNA and paraffin-embedded tissues from dogs with confirmed herpes-, adeno-, or canine parvovirus infections. Herpes-, adeno-, or canine parvovirus DNA was amplified by PCR from the corresponding positive-control specimens. Negative controls included 7 dogs with various brain disorders and produced no viral amplicons. The 22 dogs with NME, NLE, and GME were negative for viral DNA. Additional studies testing for other viruses or inherited genetic mutations are warranted to gain insight into the etiologies of NME, NLE, and GME. We discuss potential etiologies and provide a clinical and histopathologic overview of these common canine encephalitides.

Down-regulation of GAT-1 mRNA expression in the microdissected hypothalamic medial preoptic area of rat offspring exposed maternally to ethinylestradiol

Steroid hormones are powerful regulators of gene transcription in the brain and have the potential to permanently alter the structure and function of the developing brain. Steroid-mediated altered gene expression may thus be responsible for the molecular cascade for sexual differentiation. In this study, to assess effects of maternal exposure to ethinylestradiol (EE) on brain sexual differentiation of offspring, region-specific mRNA expression of two estrogen-responsive genes, gamma-aminobutyric acid transporter type 1 (GAT-1) and anti-apoptotic bcl-xL was measured in the medial preoptic area (MPOA), including sexually dimorphic nucleus (SDN), at the late stage of brain sexual differentiation in rats. Pregnant Sprague-Dawley animals were fed diets containing EE at concentrations of 0, 0.02, 0.1, and 0.5 ppm from day 15 of pregnancy to day 9 after delivery. In another group, neonates were directly injected with estradiol benzoate (EB: 10 microg/pup, sc) on postnatal day (PND) 2. The MPOA on PND 9 was microdissected from methacarn-fixed paraffin-embedded brain sections to measure mRNA levels by competitive RT-PCR, followed by plate hybridization. EE-exposure decreased GAT-1 expression dose-dependently from 0.02 ppm in females and at 0.5 ppm in males, while EB-treatment caused reduction only in females. EE-exposure did not alter Bcl-xL levels. At week 11, EE-exposed females exhibited a similar spectrum of histopathological changes in endocrine-linked organs as with EB, evident from 0.1 ppm, while in males EE-exposure did not cause histopathological alteration despite clear change with EB-treatment. Measurement of SDN-POA dimensions at week 11 revealed volume reduction in males exposed to 0.5 ppm EE or EB. The results suggest that GAT-1 expression in the developing MPOA is a sensitive measure for the level of disruption of brain sexual differentiation due to maternal dietary exposure to estrogens, despite definite reproductive abnormalities may not be detectable in males with this exposure protocol.

Do canine parvoviruses affect canine neurons? An immunohistochemical study

In cats (most of which died from panleukopenia), cerebral neurons have recently been shown to be susceptible to canine parvovirus infection. In addition to positive immunostaining and distinct in situ hybridization signals, signs of neurodegeneration were identified by histopathology, mainly in the diencephalic area. Similar histological lesions of the diencephalic regions in dogs have also attracted attention; therefore, an immunohistochemical study was initiated to determine the possible infection of canine neurons with canine parvoviruses. The study was carried out on formalin-fixed and paraffin-embedded brain tissue, with and without signs of neurodegeneration, from 40 dogs, most of them dying from parvovirus enteritis. Immunohistochemistry, using polyclonal antiserum against canine parvoviruses, was negative in all 40 cases, suggesting that, unlike cats, canine parvoviruses do not seem capable of infecting canine neurons.

JC Virus Early Protein Detection by Immunohistochemistry in Progressive Multifocal Leukoencephalopathy: A Comparative Study with In Situ Hybridization and Polymerase Chain Reaction

In situ hybridization (ISH) for JC virus (JCV) is generally applied for the diagnosis of progressive multifocal leukoencephalopathy (PML). To explore the usefulness of immunohistochemistry (IHC) for JCV early proteins, 14 paraffin-embedded postmortem brain specimens with histologic features compatible with PML were tested for the presence of JCV by means of DNA-DNA ISH with a biotinylated probe corresponding to the entire JCV genome, for JCV early proteins IHC with both PAb 2003 and anti-SV40 large T antigen monoclonal antibodies, and polymerase chain reaction (PCR) amplification of JCV virion protein 3 (VP3) and transcriptional control region (TCR) sequences. ISH was positive in 13 cases and IHC in all 14 cases, the number of IHC-positive cells generally being far in excess of ISH-positive cells. Of the 2 monoclonal antibodies used, PAb 2003 proved to be more sensitive than anti-SV40 large T antigen. Occasional neuronal nuclei were positive for JCV early proteins in 5 cases. As for PCR, VP3 was amplified in all 14 cases and TCR in 9 cases. Consequently, PAb 2003 IHC for JCV early proteins seems to be a powerful tool for viral demonstration in PML and may well become the diagnostic recourse of choice in this setting.

Transient Cerebral Ischemia Induces Aberrant Neuronal Cell Cycle Re-entry and Alzheimer's Disease-like Tauopathy in Female Rats

Aberrant mitosis occurs in many tauopathy-related neurodegenerative diseases and is believed to precede the formation of neurofibrillary tangles. In this study, we report for the first time that transient cerebral ischemia induces aberrant mitotic proteins and hyperphosphorylation of tau protein with neurofibrillary tangle-like conformational epitopes in adult female rat cortex. Following transient cerebral ischemia in rats, initiation of apoptosis precedes and is potentially integrated with subsequent aberrant mitosis and tau hyperphosphorylation. Furthermore, inhibition of mitosis-related cyclin-dependent kinases (Cdks) by roscovitine significantly reduced the hyperphosphorylation of tau. Administration of the female sex steroid and potent neuroprotective agent, 17beta-estradiol, reduced ischemia-reperfusion-induced cerebral damage and the subsequent aberrant mitosis and tauopathies. These results provide a neuropathological basis for the higher prevalence of dementia in stroke patients and support the hypothesis that apoptosis and aberrant mitosis are integrated pathological events in neurons that may play a critical role in the development of Alzheimer's disease and other tauopathy-related neuropathology.

Conditional control of human wild-type and Parkinson's disease-associated mutant alpha-synuclein in transgenic mouse brain

The presynaptic protein alpha-synuclein has been implicated in the pathophysiology of many neurodegenerative disorders, including Parkinson's Disease and dementia with Lewy bodies, collectively referred to as synucleinopathies. To advance the study of the function of alpha-synuclein in these diseases, we have used the tTA-system to generate transgenic mice, which express human wildtype or mutated [A30P] alpha-synuclein in a conditional manner. To obtain reversible expression of human alpha-synuclein in the brain of these mice, we combined the tTA-system with both the PrP promotor and CaMKIIalpha promotor. We investigated the conditional overexpression by western blot analysis and immunostaining of paraffin-embedded brains. Western blot analysis revealed that double-transgenic mice express alpha-synuclein at different levels in specific brain regions. The expression-pattern and level of human alpha-synuclein in the brain of double-transgenic mice depends on both the neuron-specific promotor and the integration site of the human alpha-synuclein construct. Histological analysis of the brain of transgenic mice showed aberrant expression of the protein in cell soma. However, Lewy body-like alpha-synuclein inclusions have not yet been identified. Administration of doxycycline down-regulates alpha-synuclein expression to basal levels in the brain of double-transgenic mice. We currently investigate if one transgenic mouse-line which highly expresses mutated [A30P] alpha-synuclein specifically in the olfactory bulb shows any loss of dopaminergic neurons and an impaired sense of smell. We also perform microarray expression analysis to gain insight in the pathomechanism underlying over-expression of human alpha-synuclein. Our conditional mouse-model may help to define the role of human alpha-synuclein in synucleinopathies and might be used to demonstrate whether neuropathological symptoms of these diseases are reversible.

Neuronal apoptosis following human brain injury

Neuronal apoptosis has been investigated in paraffin-embedded brain tissue from 103 individuals who had sustained blunt head injury by use of the in situ nick translation (ISNT) technique. In order to provide reliable data for a forensic wound age estimation, a quantitative morphometric analysis was performed. Apoptotic neuronal cells could be detected in a cortical contusion with a wound age of 45 min at the earliest and in the majority of the cases with postinfliction intervals up to 2 weeks, numerous ISNT-positive cells were found adjacent to the traumatically injured area. The presented data indicate that neuronal apoptosis peaks at about 1 day and persists for at least 22 weeks after blunt head injury. The time-dependent occurrence of apoptotic cells can contribute to a forensic timing of cortical contusions and complements other immunohistochemical parameters, especially in the early postinfliction interval.

Evidence of parvovirus replication in cerebral neurons of cats.

The correlation between parvovirus infections and lesions in the central nervous system other than cerebellar hypoplasia was studied in 100 cats. The animals were necropsied with a history of various diseases, one third showing typical clinical and pathomorphological signs of panleukopenia. In 18 cats polyclonal antiserum against canine parvovirus consistently labeled neurons mainly in diencephalic regions, whereas the cerebellar cortex remained negative in all cases. In situ hybridization with digoxigenin-labeled minus-sense RNA probes, hybridizing with monomer-replicative form DNA or mRNA, revealed positive signals in nuclei of several neurons of the brain, again excluding the cerebellum. PCR applied to formalin-fixed and paraffin-embedded brain tissue and intestinal tissues of the diseased cats and subsequent DNA sequence analysis yielded canine parvovirus type 2 (CPV-2)-like sequences in the central nervous system. Two aspects of these findings are intriguing: (i). parvoviruses appear to be capable of replicating in neurons, cells that are considered to be terminally differentiated and (ii). CPV-like viruses of the old antigenic type CPV-2 appear to be able to infect cats.

Expression of catalase mRNA and protein in adult rat brain: detection by nonradioactive in situ hybridization with signal amplification by catalyzed reporter deposition (ISH-CARD) and immunohistochemistry (IHC)/immunofluorescence (IF).

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.