Selective cholinergic denervation, independent from oxidative stress, in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by increases in amyloid-beta (Abeta) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the Abeta pathology or oxidative stress remain unclear. We therefore investigated amyloidosis-related changes in acetylcholine (ACh) and serotonin (5-HT) innervations of hippocampus and parietal cortex by quantitative choline acetyltransferase (ChAT) and 5-HT immunocytochemistry, in 6, 12/14 and 18 month-old transgenic mice carrying familial AD-linked mutations (hAPP(Sw,Ind)). Further, using manganese superoxide dismutase (MnSOD) and nitrotyrosine immunoreactivity as markers, we evaluated the relationship between oxidative stress and the ACh deficit in 18 month-old mice. Thioflavin-positive Abeta plaques were seen in both regions at all ages; they were more numerous in hippocampus and increased in number (>15-fold) and size as a function of age. A majority of plaques exhibited or were surrounded by increased MnSOD immunoreactivity, and dystrophic ACh or 5-HT axons were seen in their immediate vicinity. Counts of immunoreactive axon varicosities revealed significant decreases in ACh innervation, with a sparing of the 5-HT, even in aged mice. First apparent in hippocampus, the loss of ACh terminals was in the order of 20% at 12/14 months, and not significantly greater (26%) at 18 months. In parietal cortex, the ACh denervation was significant at 18 months only, averaging 24% across the different layers. Despite increased perivascular MnSOD immunoreactivity, there was no evidence of dystrophic ACh varicosities or their accentuated loss in the perivascular area. Moreover, there was virtually no sign of tyrosine nitration in ChAT nerve terminals or neuronal cell bodies. These data suggest that aggregated Abeta exerts an early, non-selective and focal neurotoxic effect on both ACh and 5-HT axons, but that a selective, plaque- and oxidative stress-independent diffuse cholinotoxicity, most likely caused by soluble Abeta assemblies, is responsible for the hippocampal and cortical ACh denervation.

Dominant negative effects of apolipoprotein E4 revealed in transgenic models of neurodegenerative disease.

Apolipoprotein E fulfills fundamental functions in lipid transport and neural tissue repair after injury.(6,8) Its three most common isoforms (E2, E3, and E4) are critical determinants of diverse human diseases, including major cardiovascular and neurodegenerative disorders.(8,14) Apolipoprotein E4 is associated with an increased risk for Alzheimer's disease(3,5) and poor clinical outcome after head injury or stroke.(11,16) The precise role of apolipoprotein E4 in these conditions remains unknown. To characterize the effects of human apolipoprotein E isoforms in vivo, we analysed transgenic Apoe knockout mice that express apolipoprotein E3 or E4 or both in the brain. Hemizygous and homozygous apolipoprotein E3 mice were protected against age-related and excitotoxin-induced neurodegeneration, whereas apolipoprotein E4 mice were not. Apolipoprotein E3/E4 bigenic mice were as susceptible to neurodegeneration as apolipoprotein E4 singly-transgenic mice. At eight months of age neurodegeneration was more severe in homozygous than in hemizygous apolipoprotein E4 mice consistent with a dose effect. Thus, apolipoprotein E4 is not only less neuroprotective than apolipoprotein E3 but also acts as a dominant negative factor that interferes with the beneficial function of apolipoprotein E3. The inhibition of this apolipoprotein E4 activity may be critical for the prevention and treatment of neurodegeneration in APOE varepsilon4 carriers.

Elimination of the class A scavenger receptor does not affect amyloid plaque formation or neurodegeneration in transgenic mice expressing human amyloid protein precursors.

The class A scavenger receptor (SR) is expressed on reactive microglia surrounding cerebral amyloid plaques in Alzheimer's disease (AD). Interactions between the SR and amyloid beta peptides (Abeta) in microglial cultures elicit phagocytosis of Abeta aggregates and release of neurotoxins. To assess the role of the SR in amyloid clearance and Abeta-associated neurodegeneration in vivo, we used the platelet-derived growth factor promoter to express human amyloid protein precursors (hAPPs) in neurons of transgenic mice. With increasing age, hAPP mice develop AD-like amyloid plaques. We bred heterozygous hAPP (hAPP(+/-)) mice that were wild type for SR (SR(+/+)) with SR knockout (SR(-/-)) mice. Crosses among the resulting hAPP(+/-)SR(+/-) offspring yielded hAPP(+/-) and hAPP(-/-) littermates that were SR(+/+) or SR(-/-). These second-generation mice were analyzed at 6 and 12 months of age for extent of cerebral amyloid deposition and loss of synaptophysin-immunoreactive presynaptic terminals. hAPP(-/-)SR(-/-) mice showed no lack of SR expression, plaque formation, or synaptic degeneration, indicating that lack of SR expression does not result in significant accumulation of endogenous amyloidogenic or neurotoxic factors. In hAPP(+/-) mice, ablation of SR expression did not alter number, extent, distribution, or age-dependent accumulation of plaques; nor did it affect synaptic degeneration. Our results do not support a critical pathogenic role for microglial SR expression in neurodegenerative alterations associated with cerebral beta amyloidosis.

Cultured rat microglia express functional beta-chemokine receptors.

We have investigated the functional expression of the beta-chemokine receptors CCR1 to 5 in cultured rat microglia. RT-PCR analysis revealed constitutive expression of CCR1, CCR2 and CCR5 mRNA. The beta-chemokines MCP-1 (1-30 nM) as well as RANTES and MIP-1alpha (100-1000 nM) evoked calcium transients in control and LPS-treated microglia. Whereas, the response to MCP-1 was dependent on extracellular calcium the response to RANTES was not. The effect of MCP-1 but not that of RANTES was inhibited by the calcium-induced calcium release inhibitor ryanodine. Calcium responses to MCP-1- and RANTES were observed in distinct populations of microglia.

Expression of human apolipoprotein E3 or E4 in the brains of Apoe-/- mice: isoform-specific effects on neurodegeneration.

Apolipoprotein (apo) E isoforms are key determinants of susceptibility to Alzheimer's disease. The apoE4 isoform is the major known genetic risk factor for this disease and is also associated with poor outcome after acute head trauma or stroke. To test the hypothesis that apoE3, but not apoE4, protects against age-related and excitotoxin-induced neurodegeneration, we analyzed apoE knockout (Apoe-/-) mice expressing similar levels of human apoE3 or apoE4 in the brain under control of the neuron-specific enolase promoter. Neuronal apoE expression was widespread in the brains of these mice. Kainic acid-challenged wild-type or Apoe-/- mice had a significant loss of synaptophysin-positive presynaptic terminals and microtubule-associated protein 2-positive neuronal dendrites in the neocortex and hippocampus, and a disruption of neurofilament-positive axons in the hippocampus. Expression of apoE3, but not of apoE4, protected against this excitotoxin-induced neuronal damage. ApoE3, but not apoE4, also protected against the age-dependent neurodegeneration seen in Apoe-/- mice. These differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of Alzheimer's disease and to the poor outcome after head trauma and stroke associated with apoE4 in humans.

Isoform-specific effects of human apolipoprotein E on brain function revealed in ApoE knockout mice: increased susceptibility of females.

Apolipoprotein E (apoE) mediates the redistribution of lipids among cells and is expressed at highest levels in brain and liver. Human apoE exists in three major isoforms encoded by distinct alleles (epsilon2, epsilon3, and epsilon4). Compared with APOE epsilon2 and epsilon3, APOE epsilon4 increases the risk of cognitive impairments, lowers the age of onset of Alzheimer's disease (AD), and decreases the response to AD treatments. Besides age, inheritance of the APOE epsilon4 allele is the most important known risk factor for the development of sporadic AD, the most common form of this illness. Although numerous hypotheses have been advanced, it remains unclear how APOE epsilon4 might affect cognition and increase AD risk. To assess the effects of distinct human apoE isoforms on the brain, we have used the neuron-specific enolase (NSE) promoter to express human apoE3 or apoE4 at similar levels in neurons of transgenic mice lacking endogenous mouse apoE. Compared with NSE-apoE3 mice and wild-type controls, NSE-apoE4 mice showed impairments in learning a water maze task and in vertical exploratory behavior that increased with age and were seen primarily in females. These findings demonstrate that human apoE isoforms have differential effects on brain function in vivo and that the susceptibility to apoE4-induced deficits is critically influenced by age and gender. These results could be pertinent to cognitive impairments observed in human APOE epsilon4 carriers. NSE-apoE mice and similar models may facilitate the preclinical assessment of treatments for apoE-related cognitive deficits.

Novel role of human CD4 molecule identified in neurodegeneration.

The human CD4 molecule (hCD4) is expressed on T lymphocytes and macrophages and acts as a key component of the cellular receptor for HIV. At baseline, hCD4 transgenic mice expressed hCD4 on microglia, the resident mononuclear phagocytes of the brain, and showed no neuronal damage. Activation of brain microglia by peripheral immune challenges elicited neurodegeneration in hCD4 mice but not in nontransgenic controls. In post-mortem brain tissues from AIDS patients with opportunistic infections, but without typical HIV encephalitis, hCD4 expression correlated with neurodegeneration. We conclude that hCD4 may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system.

Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and peripheral lipopolysaccharide administration.

Increasing evidence indicates a key role of chemoattractant cytokines in the accumulation of leukocytes in the central nervous system (CNS) during the course of inflammatory processes. Monocyte chemoattractant protein (MCP-1/JE), a member of the beta-chemokine (C-C chemokine) family, functions as a potent chemoattractant and activator for monocytes. We have investigated the induction of MCP-1 mRNA using in situ hybridization histochemistry (ISH) and characterized its cellular source by combination of ISH and immunocytochemistry in ischemic rat brains as well as in brains of endotoxin-treated rats. Our results show that 6 h-2 d after middle cerebral artery occlusion (MCAO), MCP-1 mRNA is present in astrocytes surrounding the ischemic tissue (penumbra). At later time points (after 4 d), MCP-1 mRNA is found in macrophages and reactive microglia in the infarcted tissue. Peripheral administration of the bacterial lipopolysaccharide (LPS) induced MCP-1 mRNA throughout the brain in a time-dependent manner (1 h-1 d, peak of expression 6-8 h) and was found in astrocytes. In summary, we have found expression of MCP-1 in (a) astrocytes and to a lesser extent in macrophages/reactive microglia after MCA-occlusion and in (b) astrocytes after peripheral administration of LPS. These findings support that MCP-1 is involved in the CNS response to acute trauma or infection and thus may play a key role in inflammatory processes of the brain.

Low malignant potential ovarian tumours: a review of 175 consecutive cases.

Epithelial ovarian tumours of low malignant potential (LMP) are known to have a generally good prognosis, although there is not universal agreement on all aspects of treatment. We report a series of 175 patients with LMP ovarian tumours referred to the Queensland Centre for Gynaecological Cancer between January, 1982 and December, 1993. Stage I disease accounted for 142 cases, with only 1 patient dead from disease at 293 months. Twenty nine patients in this group had conservative surgery with 1 recurrence only (in the contralateral ovary) giving a recurrence rate of 3.5%. Survival and treatment data for other stages are presented, and the current literature reviewed. It is suggested that early stage disease may be treated conservatively depending upon the patient's desire to retain reproductive capacity. While adjuvant therapy is not recommended, long-term follow-up is indicated. More advanced disease should be debulked to the smallest practical volume. The role of lymphadenectomy has been questioned, as survival has not been shown to be affected by treatment decisions made as a result of knowing the lymph node status. Whilst some centres give platinum-based adjuvant therapy, the evidence that it is beneficial is not supported by any prospective randomized trials.

Lipopolysaccharide induces expression of tumour necrosis factor alpha in rat brain: inhibition by methylprednisolone and by rolipram.

1. We have investigated the effects of the phosphodiesterase (PDE) type IV inhibitor rolipram and of the glucocorticoid methylprednisolone on the induction of tumour necrosis factor alpha (TNF-alpha) mRNA and protein in brains of rats after peripheral administration of lipopolysaccharide (LPS). 2. After intravenous administration of LPS, a similar time-dependent induction of both TNF-alpha mRNA and protein was observed in rat brain. Peak mRNA and protein levels were found 7 h after administration of LPS. 3. In situ hybridization experiments with a specific antisense TNF-alpha riboprobe suggested that the cells responsible for TNF-alpha production in the brain were microglia. 4. Intraperitoneal administration of methylprednisolone inhibited the induction of TNF-alpha protein in a dose-dependent manner. A maximal inhibition of TNF-alpha protein production by 42.9+/-10.2% was observed at a dose regimen consisting of two injections of each 30 mg kg(-1) methylprednisolone. 5. Intraperitoneal administration of rolipram also inhibited the induction of TNF-alpha protein in a dose-dependent manner. The maximal inhibition of TNF-alpha protein production was 96.1+/-12.2% and was observed at a dose regimen of three separate injections of each 3 mg kg(-1) rolipram. 6. In situ hybridization experiments showed that the level of TNF-alpha mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of methylprednisolone (2 x 15 mg kg(-1)) and of rolipram (3 x 3 mg kg(-1)). 7. We suggest that peripheral administration of LPS induces a time-dependent expression of TNF-alpha in rat brain, presumably in microglial cells, and that methylprednisolone and rolipram inhibit LPS-induced expression of TNF-alpha in these cells via a decrease of TNF-alpha mRNA stability and/or TNF-alpha gene transcription.

An analysis of HIV-1-associated inflammatory products in brain tissue of humans and SCID mice with HIV-1 encephalitis.

The human immunodeficiency virus type 1 (HIV)-associated dementia complex (ADC) is a neuroimmunological disorder fueled by viral replication in mononuclear phagocytes (MP) (brain macrophages and microglia). The elucidation of MP inflammatory factors involved in neurological dysfunction is pivotal for unraveling pathogenic mechanisms and in developing new therapies for this disease. Recent advances in animal model systems for ADC and its associated encephalitis have provided important insights into how virus-infected macrophages cause brain injury. Indeed, the stereotactic inoculation of HIV infected monocytes into the basal ganglia/cortex of mice with severe combined immunodeficiency disease (SCID) results in pathological features similar to those of human HIV-1 encephalitis (HIVE). We used this SCID model to study the roles of macrophage secretory factors in HIVE. The expression of interleukin-1 (IL-1 beta, IL-6, IL-10), tumor necrosis factors-alpha (TNF alpha), vascular endothelial growth factor (VEGF), and adhesion molecules (E-selectin, intracellular cell adhesion molecule (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)) in encephalitic brains of mice and humans was evaluated by semi-quantitative polymerase chain reaction (PCR). In SCID mice with HIVE, human and mouse TNF alpha, and mouse IL-6, VEGF, VCAM-1 and E-selectin were expressed at high levels. These results paralleled, to a great extent, those in HIVE brain tissues. Laser scanning confocal microscopy performed to assess the associated neuronal damage showed that microtubule associated protein-2 (MAP-2) immunoreactive dendrites were significantly reduced in both the ipsilateral and contralateral hemispheres of encephalitic mice. These results demonstrate the importance of macrophage inflammatory products in the pathogenesis of HIVE and further validates this model of viral encephalitis in SCID mice.

Peripheral administration of lipopolysaccharide induces activation of microglial cells in rat brain.

Using immunocytochemistry with monoclonal antibodies against surface immunomolecules and Griffonia simplicifolia lectin histochemistry, the microglial cell reaction in rat brain was studied after intravenous injection of lipopolysaccharide (LPS). Activation of microglial cells throughout the brain became apparent within hours and peaked at 8-24 h following administration of 1, 2.5 and 5 mg/kg LPS. High doses of LPS (2.5 and 5 mg/kg) induced a morphological transition of resting ramified microglia to round, macrophage-like cells in the anterior hypothalamus, thalamus and the brainstem. After injection of 1 mg/kg LPS, this morphological transition was only detected in the brainstem. Microglial cell reactivity gradually returned to control levels within 7 days after LPS administration. Furthermore, LPS induced enhanced expression of MHC class II by microglial cells. Maximal up-regulation of MHC class II Ia-antigen was found 3 days following injection of LPS, and only a few highly Ia immunoreactive cells were detectable 7 days following injection of LPS. Despite the presence of highly activated microglial cells in the rat brain, no signs of tissue damage were observed at any time point after injection of LPS examined. In addition to the activation of microglial cells, intravenous injection of LPS induced accumulations of macrophages in blood vessels of the choroid plexus and the brain, but no disruption of vessels with subsequent invasion of parenchyma by blood macrophages was detected. Our data demonstrate that a peripheral immune challenge leads to a high and transitory activation of microglial cells in the brain which could possibly contribute to the pathology of infections and septic shock.

Expression of tumor necrosis factor alpha after focal cerebral ischaemia in the rat.

Induction of tumor necrosis factor alpha was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for in situ hybridization histochemistry, cells positive for tumor necrosis factor alpha messenger RNA were detected within 30 min in the brain regions known to be necrotic within one to two days after onset of ischaemia. Their number increased over a time period of 1-8 h and then declined. Only a few tumor necrosis factor alpha messenger RNA positive cells could be detected four days after the onset of ischaemia. Reverse-transcription polymerase chain reaction experiments showed that maximal increase of tumor necrosis factor alpha messenger RNA level in the ischaemic brain hemisphere occurred 3 h after occlusion of the middle cerebral artery. Immunocytochemical experiments using an anti-tumor necrosis factor alpha antibody showed the presence of tumor necrosis factor alpha immunopositive cells as early as 30 min after occlusion of the middle cerebral artery in the same brain regions where tumor necrosis factor alpha messenger RNA positive cells were detected. Tumor necrosis factor alpha positive cells were highly abundant in the infarcted brain 8-24 h, but only few of them were detectable four days after the onset of ischaemia. Specificity of the anti-tumor necrosis factor alpha antibody and of the induction of tumor necrosis factor alpha protein was confirmed by western blot analysis. Tumor necrosis factor alpha messenger RNA- and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. According to their morphology, tumor necrosis factor alpha-positive cells could be identified as microglial cells and macrophages at different states of activation. This assumption was further confirmed by double-labeling studies using the isolectin B4 from Griffonia simplicifolia, a specific microglial/macrophage cell marker. These results demonstrate that expression of tumor necrosis factor alpha is part of an intrinsic inflammatory reaction of the brain after ischaemia.

Distribution of the mRNA for protein phosphatase T in rat brain.

We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by in situ hybridization histochemistry. PPT mRNA was found to be differentially expressed throughout the rat brain. Highest transcript levels were found in specific neuronal populations (hippocampus, piriform cortex, taenia tecta, medial habenula, granular cell layer of the cerebellum) as well as in the choroid plexus of the third and lateral ventricles. In contrast, expression levels in some brain areas, e.g., caudate putamen and white matter, were beyond the detection limit of in situ hybridization. The pattern of expression of PPT in rat brain differs from that of other protein serine/threonine phosphatases and may reflect specific functions of this phosphatase.

Cloning of rat interleukin-3 receptor beta-subunit from cultured microglia and its mRNA expression in vivo.

The high-affinity receptors for interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of a ligand binding (alpha-) and a transducing (beta-) subunit. Two distinct transducing subunits (clones AIC2A and AIC2B) have been cloned from mouse, whereas in humans, only one (common) beta-subunit (beta c) has been found. A PCR-based cloning strategy was used to obtain a full-length cDNA sequence from rat microglia including 5'-untranslated regions. Sequence analysis revealed a number of features indicative of the presence of only one beta-subunit in the rat. Most likely, the new rIL-3R beta cDNA is the rat equivalent of human respective murine (AIC2B) beta c subunits. Regulation of rIL-3R beta mRNA expression was investigated in cultured microglia and in vivo. Purified microglia expressed significant amounts of rIL-3R beta mRNA. Addition of lipopolysaccharide (LPS) resulted in a marked upregulation of rIL-3R beta mRNA within approximately 4 hr. No downregulation was observed within 1 week's treatment. No rIL-3R beta mRNA was detectable in normal rat brain. However, 3 hr after a single injection of LPS into the tail vein of a rat, a marked induction of receptor mRNA occurred in a variety of brain regions. Transcriptional rates subsided significantly after 24 hr. rIL-3R beta mRNA was visualized by in situ hybridizations with cRNA antisense probes in ramified cells formerly characterized as microglial cells. rIL-3R beta mRNA was also induced in rat brain after occlusion of middle cerebral artery (MCAO).(ABSTRACT TRUNCATED AT 250 WORDS)

Fibroid arising in a müllerian duct remnant and presenting as a pelvic mass in a patient with vaginal agenesis: an unusual finding.

We report a case of vaginal agenesis associated with a large fibroid arising from a Müllerian duct remnant. Vaginal agenesis is rare and infrequently encountered by gynaecologists. Its association with a pelvic mass is even more uncommon, and a medline search by the authors has failed to uncover another reported case. We provide the clinical details of this case and discuss the differential diagnoses.

Distribution of calcineurin A isoenzyme mRNAs in rat thymus and kidney.

The distribution of the mRNAs encoding the different isoforms of the catalytic subunit (A subunit) of calcineurin has been investigated in rat thymus and kidney using in situ hybridization histochemistry with specific antisense oligonucleotide probes. In the thymus, the mRNAs of the A beta isoforms were the predominant transcripts and showed very intense hybridization signals in the cortical areas. The A alpha mRNAs were expressed at low levels. A beta 2 mRNA was expressed at higher levels than A beta 3 mRNA, but no difference could be detected between the expression levels of A alpha 1 and A alpha 2. In the kidney, highest calcineurin A mRNA hybridization signals were found in the medulla. Signal intensities of A alpha mRNAs were comparable to those of A beta mRNAs. A alpha 1 mRNA level was extremely weak, and A beta 2 mRNA expression was slightly higher than A beta 3 mRNA expression. A tissue-specific distribution pattern of the alternatively spliced isoforms of calcineurin A, as suggested by these preliminary data from thymus and kidney, may be critical in understanding the toxic side-effects associated with the use of the immunosuppressive, calcineurin-inhibiting compounds cyclosporin A and FK506.

Peripheral lipopolysaccharide stimulation induces interleukin-1 beta messenger RNA in rat brain microglial cells.

The inflammatory cytokine interleukin-1 acts as an endogenous pyrogen in organisms affected by infectious diseases and has been shown to influence the activity of the central nervous system. Using in situ hybridization histochemistry, we have examined the cellular source of interleukin-1 beta in rat brain after peripheral stimulation with the bacterial lipopolysaccharide, a potent inducer of interleukin-1. Whereas no interleukin-1 beta messenger RNA could be detected in brains in unstimulated rats, lipopolysaccharide induced a transient, high and widespread expression of interleukin-1 beta messenger RNA in the entire brain. The highest levels of interleukin-1 beta messenger RNA were observed 6-8 h after stimulation with lipopolysaccharide. Using a combination of non-radioactive in situ hybridization and immunocytochemistry with the microglial-specific antibody OX-42, interleukin-1 beta messenger RNA-positive cells could be identified as microglia. We conclude that brain microglial cells are the major source of interleukin-1 beta messenger RNA after peripheral administration of lipopolysaccharide.