HIV encephalopathy: glial activation and hippocampal neuronal apoptosis, but limited neural repair.

OBJECTIVES: HIV infection affects the central nervous system (CNS), frequently causing cognitive impairment. Hippocampal injury impedes the ability to transfer information into memory. Therefore, we aimed to examine neuronal injury and repair in the hippocampal formation in HIV encephalopathy. METHODS: We compared neuropathological findings in 14 autopsy cases after death from systemic complications of HIV infection and in 15 age-matched HIV-negative control cases after sudden death from nonneurological causes using immunohistochemistry. RESULTS: The density of apoptotic granule cells in the dentate gyrus was higher in HIV-infected than in control cases (P = 0.048). Proliferation of neural progenitor cells in the dentate gyrus was increased in HIV infection (P = 0.028), whereas the density of recently generated TUC-4 [TOAD (turned on after division)/Ulip/CRMP family 4]-expressing neurons in this region was not significantly elevated in HIV-infected cases (P = 0.13). HIV infection caused microglial activation and astrocytosis in the neocortex and hippocampal formation. Conversely, we were unable to detect more pronounced axonal injury in HIV-infected than in control cases. CONCLUSIONS: As in other infections involving the CNS, apoptosis of hippocampal neurons accompanied by microglial activation and astrocytosis is a prominent feature of HIV encephalopathy. The regenerative potential, assessed using the density of young neurons in the hippocampal dentate gyrus, in HIV infection appears to be lower than in acute bacterial meningitis and septic encephalitis.

Septic metastatic encephalitis: coexistence of brain damage and repair.

AIMS: Septic metastatic encephalitis (SME) arises from systemic bacterial infections and is a severe complication of sepsis with a high mortality. In this study, we examined the neuropathological findings in humans suffering from SME including white matter pathology and proliferation of neural precursor cells in the hippocampal dentate gyrus. METHODS: The brains of 10 autopsy cases with SME and 10 control cases after sudden death from non-neurological causes were studied by means of immunohistochemistry. RESULTS: We found diffuse axonal injury and demyelination in the frontal cortex (P = 0.01) as well as increased numbers of recently generated TUC-4 expressing neurones in the hippocampal dentate gyrus in SME cases (P = 0.01). The median density of apoptotic granule cells in the dentate gyrus also was higher in SME cases, the difference, however, failed to reach statistical significance (P = 0.25). CONCLUSION: The coexistence of degenerative processes predominantly in the neocortex and regenerative activity in the hippocampal formation known from bacterial meningitis also characterizes the pathology of SME.

S100B in the cerebrospinal fluid--a marker for glial damage in the rabbit model of pneumococcal meningitis.

The rabbit model provides an important experimental setting for the evaluation of antibiotic agents against pneumococcal meningitis. One of the primary targets of this model is the study of neuronal and glial cell damage in bacterial meningitis. The aim of this investigation was to evaluate whether a significant increase of S100B in the cerebrospinal fluid (CSF) as an indicator of white matter damage could be observed in this meningitis model. Seven rabbits were infected intracisternally with S. pneumoniae, and CSF S100B concentrations were examined serially before infection, at 12h, 14h, 17h, 20h, and at 24h after infection. The course of CSF S100B increase and its relation to other parameters of brain tissue destruction and CSF inflammation were measured. Axonal damage was visualized by amyloid precursor protein (APP) immunostaining and demyelination by Luxol Fast Blue/Periodic Acid Schiff (LFB-PAS) stain. In each animal, we observed a distinct rise in S100B concentration in the CSF due to pneumococcal meningitis. We conclude that the CSF concentration of the glial S100B protein can be used as an additional parameter for future interventional studies focusing on glial cell damage in the rabbit model of bacterial meningitis.

Evidence for frequent focal and diffuse acute axonal injury in human bacterial meningitis.

AIMS: We aimed at quantifying acute axonal injury in victims of bacterial meningitis. METHODS: The brains of 26 autopsies with bacterial meningitis and of 10 control cases were studied by histology and quantitative immunohistochemistry for amyloid-beta precursor protein (APP). RESULTS: Mild to severe axonal injury in the white matter was present in 25 of 26 victims of meningitis. The area of axonal damage ranged from 0.0% to 1.38% (median = 0.08%, mean = 0.36%) of the total area studied in each individual case. In 4 of 10 age- and sex-matched control brains small areas also stained for APP (p = 0.0007). Axonal injury in meningitis was most prominent in the basal ganglia and pons, followed by the hippocampal formation, neocortex and the cervical spinal cord. The cerebellum was least affected. CONCLUSION: Axonal injury is a frequent complication of bacterial meningitis probably contributing to long-term sequelae in survivors.

Expression of death-related proteins in dentate granule cells in human bacterial meningitis.

Neuronal apoptosis in the dentate gyrus has been observed in animal models of bacterial meningitis and in humans dying in the course of the disease. To evaluate the mechanisms of neuronal cell death, hippocampal sections of 20 patients dying from bacterial meningitis were investigated by immunohistochemistry using antibodies against the proform of caspase-3 and the active enzyme, bcl-2, bax and p53. In the dentate granule cell layer, the median density of neurons with an apoptotic morphology was 7.6/mm2 (0-15.6/mm2). The median density of immunoreactive neurons was 2.3/mm2 (procaspase-3), 0.9/mm2 (activated caspase-3), 1.8/mm2 (bcl-2), 1.1/mm2 (bax) and 0.4/mm2 (p53). 80% of neurons immunoreactive for active caspase-3 had an apoptotic morphology, whereas only 10% of all procaspase-3 stained neurons showed signs of apoptosis. Apoptotic cell death is present in humans dying in the course of bacterial meningitis in the dentate gyrus of the Formatio hippocampi. Neuronal expression of caspase-3, bcl-2 and bax suggests an involvement of these proteins in neuronal death.

Transcriptional regulation of caspases in experimental pneumococcal meningitis.

Apoptosis and necrosis in brain account for neurological sequelae in survivors of bacterial meningitis. In meningitis, several mechanisms may trigger death pathways leading to activation of transcription factors regulating caspases mRNA synthesis. Therefore, we used a multiprobe RNA protection assay (RPA) to examine the expression of 9 caspase-mRNA in the course of experimental Streptococcus pneumoniae meningitis in mouse brain. Caspase-6, -7 and -11 mRNA were elevated 6 hours after infection. 12 hours after infection caspases-1, -2, -8 and -12 mRNA rose. Caspase-14 mRNA was elevated 18 h and caspase-3 mRNA 24 h after infection. In situ hybridization detected caspases-3, -8, -11 and -12 mRNA in neurons of the hippocampal formation and neocortex. Development of sepsis was paralleled by increased transcription of caspases mRNA in the spleen. In TNFalpha-deficient mice all caspases examined were less upregulated, in TNF-receptor 1/2 knockout mice caspases-1, -2, -7, -11 and -14 mRNA were increased compared to infected control animals. In caspase-1 deficient mice, caspases-11, and -12 mRNA levels did not rise in meningitis indicating the necessity of caspase-1 activating these caspases. Hippocampal formations of newborn mice incubated with heat-inactivated S. pneumoniae R6 showed upregulation of caspase-1, -3, -11 and -12 mRNA. These observations suggest a tightly regulated caspases network at the transcriptional level in addition to the known cascade at the protein level.

Follistatin (FS) in human cerebrospinal fluid and regulation of FS expression in a mouse model of meningitis.

OBJECTIVE: Follistatin (FS) is the specific binding protein of activin and expression of both factors is regulated by inflammatory agents. Therefore, FS concentrations were determined in cerebrospinal fluid (CSF) of patients with bacterial and viral meningitis or multiple sclerosis (MS), as well as in the CSF of patients without meningial inflammation or autoimmune diseases. Furthermore, a mouse pneumococcal meningitis model was used to localise the cellular sources of FS in brains of normal and meningitic mice. METHODS: FS concentrations in CSF were determined by ELISA; FS in mice was localised by in situ hybridisation and immunohistochemistry. RESULTS: FS concentrations were > or =0.4 microg/l in 22 of 66 CSF samples of meningitis patients versus 2 of 27 CSF samples from patients with multiple sclerosis (P<0.05) and 2 of 41 CSF specimen from patients without neuroinflammatory diseases (P<0.01). In the CSF of patients with meningitis, the concentration of FS was correlated with total protein (P<0.005) and lactate concentrations (P<0.05), but not with leukocyte counts, interval between onset of disease and CSF analysis, or clinical outcome. The CSF-to-serum ratios of FS and albumin also correlated significantly (P<0.0005). In some patients with meningitis the CSF-to-serum ratios suggested that the elevated FS in CSF did not originate from serum alone. FS was localised in mice brains to neurones of the hippocampus, dentate gyrus, neocortex, and to the choroid plexus. Analyses of brains and other organs from uninfected and infected animals sacrificed 6-36 h after infection did not reveal any obvious differences in the distribution and intensity of FS mRNA and protein expression. CONCLUSIONS: The concentration of FS in humans is elevated during meningitis. In some patients the increase is caused by a release of FS from brain into CSF. Data from the mouse meningitis model suggest that increased CSF concentrations of FS in meningitis appear not to be accompanied by an elevated number of cells containing FS mRNA or protein in the brain.

Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation.

Damage to axons is taken as a key factor of disability in multiple sclerosis, but its pathogenesis is largely unknown. Axonal injury is believed to occur as a consequence of demyelination and was recently shown to be a feature even of the early disease stages. The present study was aimed at characterizing the association of axonal injury and histopathological hallmarks of multiple sclerosis such as demyelination, cellular infiltration and expression of inflammatory mediators. Therefore, axon reduction and signs of acute axonal damage were quantified in early lesion development of chronic multiple sclerosis and correlated with demyelinating activity and inflammation. Patients with secondary progressive multiple sclerosis revealed the most pronounced axonal injury, whereas primary progressive multiple sclerosis patients surprisingly showed relatively little acute axonal injury. Acute axonal damage, as defined by the accumulation of amyloid precursor protein (APP), was found to occur not only in active demyelinating but also in remyelinating and inactive demyelinated lesions with a large inter-individual variability. Only few remyelinating lesions were adjacent to areas of active demyelination. In this minority of lesions, axonal damage may have originated from the neighbourhood. APP expression in damaged axons correlated with the number of macrophages and CD8-positive T lymphocytes within the lesions, but not with the expression of tumour necrosis factor-alpha (TNF-alpha) or inducible nitric oxide synthase (iNOS). Axonal injury is therefore, at least in part, independent of demyelinating activity, and its pathogenesis may be different from demyelination. This has major implications for therapeutic strategies, which aim at preventing both demyelination and axonal loss.

Tumour necrosis factor alpha mRNA expression in early multiple sclerosis lesions: correlation with demyelinating activity and oligodendrocyte pathology.

The precise role of tumour necrosis factor alpha (TNFalpha) in multiple sclerosis (MS) is still controversial. Most findings from the animal model experimental allergic encephalomyelitis have yet to be confirmed in multiple sclerosis. The aim of this study was to define the significance of TNFalpha with respect to the hallmark of MS, that is demyelination. Therefore, 78 lesion areas from diagnostic brain biopsies of 32 patients were analysed. Lesion demyelinating activity was classified by the presence of myelin degradation products in macrophages and macrophage activation markers. Non-radioactive in situ hybridisation was carried out to detect TNFalpha mRNA expressing cells. DNA fragmentation was visualised by TdT-mediated X-dUTP nick end labeling. A significantly higher number of cells expressed TNFalpha mRNA in active demyelinating lesions than in inactive or remyelinating lesions irrespective of the extent of the inflammatory infiltrate. TNFalpha mRNA expression correlated with the appearance of DNA fragmentation in T lymphocytes and oligodendrocytes within the lesions. In the periplaque white matter, expression of TNFalpha mRNA negatively correlated with oligodendrocyte numbers. These data support previous findings from animal models and in vitro experiments. Although not proving, the current study strongly suggests a pathogenic role of TNFalpha in demyelination in human multiple sclerosis and gives further support for TNFalpha-directed therapeutic strategies.

Lesion development in Marburg's type of acute multiple sclerosis: from inflammation to demyelination.

We report a patient who suffered from acute inflammatory CNS demyelination and underwent two consecutive diagnostic stereotactic brain biopsies during the early disease course. The first lesion was drawn 33 days after the onset of disseminated neurological symptoms. Macrophages and T lymphocytes diffusely infiltrated small vessel walls and the white matter. mRNA for tumor necrosis factor alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) was abundantly expressed. Myelin sheaths were entirely preserved. The second biopsy 76 days later showed confluent demyelinating lesions with a diffuse infiltration of macrophages that were positive for myelin debris, activation markers and TNFalpha and iNOS mRNA. IgG and C9neo deposits were found along myelin sheaths. The patient had received intravenous immunoglobulins (IVIG) prior to biopsy. Findings from this single patient affirm that demyelination follows the migration of inflammatory cells from the circulation into the white matter with subsequent inflammation and demyelination. Inflammation alone may be sufficient to cause significant clinical deficits without demyelination. Inflammatory mediators such as TNFalpha and NO are involved at very early stages in the pathogenetic process. IVIG treatment may lead to the deposition of immunoglobulins and to the activation of the complement cascade, but the clinical relevance of this particular finding remains uncertain.

Oligodendrocyte and axon pathology in clinically silent multiple sclerosis lesions.

Oligodendrocyte and axon pathology was studied in 11 autopsy cases of clinically silent multiple sclerosis. A total of 54 lesions, either demyelinated or late remyelinated, were distributed through the whole brain and spinal cord with 39% of the lesions located in periventricular areas. Determination of axon density revealed an average reduction of 64% and 59% in demyelinated and remyelinated lesions with an extreme variation between different plaques and cases. Oligodendrocytes were identified by immunocytochemistry for myelin oligodendrocyte glycoprotein (MOG) and in situ hybridization for proteolipid protein (PLP) mRNA. Oligodendrocytes were almost completely lost in demyelinated lesions; remyelinated lesions revealed preservation of a considerable number of oligodendrocytes within the lesions. At the border between plaques and the periplaque white matter, similar oligodendrocyte numbers as in remyelinated lesions were found. Different factors including lesion site, axonal preservation and remyelination may thus contribute to the clinical nonappearance of multiple sclerosis lesions.

Identification of macrophage populations expressing tumor necrosis factor-alpha mRNA in acute multiple sclerosis.

Macrophages are involved in central nervous system (CNS) demyelination in multiple sclerosis (MS) and other demyelinating diseases. Macrophages seen in MS lesions form a heterogeneous population with respect to their stage of activation and differentiation. We have analyzed macrophages from active demyelinating lesions of a patient who died from fulminant MS of Marburg's type to define the functional heterogeneity of different macrophage populations in acute demyelination. We examined tumor necrosis factor-alpha (TNF-alpha) mRNA expression in macrophages defined by different activation markers. The majority of TNF-alpha mRNA-positive cells were macrophages positive for the pan-macrophage marker KiM1P. A subgroup of TNF-alpha mRNA-positive macrophages was stained by the early activation marker MRP14. In contrast, macrophages positive for the acute activation marker 27E10 were entirely negative for TNF-alpha mRNA. In conclusion, macrophages in acute demyelinating CNS lesions are heterogeneous as shown by staining for different activation markers. This heterogeneity is also of functional relevance as certain subpopulations are involved in TNF-alpha mRNA expression, while others are not. This may be important for directing therapeutic strategies against well-defined pathogenic macrophage populations.

DNA-protein binding studies in the 5' flanking region of rat proacrosin gene which is transcribed in diploid germ cells.

The proacrosin gene is transcribed in diploid spermatogenic cells and translated in haploid round spermatids. In order to evaluate sequences which are involved in proacrosin gene transcription, DNA-protein interactions were analyzed in 1.2 kb of the 5'flanking region of the rat gene. 13 protein binding sites were identified by DNase I footprinting using nuclear extracts from rat testis and brain, respectively. Five footprints (F1, F3, F7, TS2, TS3) which suggest an interaction with testis specific nuclear factors were further examined by gel retardation assays. Three testis specific binding sites (F1, F7, TS2, located 472bp, 697bp and 1004bp upstream of ATG, respectively) could be identified with both methods. The binding site F1 contains a motif which is similar to a testis specific footprint found in mouse protamine 1 gene. The nucleotide sequence of F7 contains the recognition motif of an isoform of the transcription factor GATA1, which is expressed in testis. Furthermore F1 and F7 are located in that part of the 5'flanking region of the proacrosin gene, which can direct proacrosin gene expression in germ cells of male transgenic mice.