Oral and subcutaneous administration of the glycosaminoglycan C3 attenuates Abeta(25-35)-induced abnormal tau protein immunoreactivity in rat brain.

High molecular weight glycosaminoglycans (GAG) and proteoglycans (PG) affect pathological changes of the brain in Alzheimer's disease (AD). PG stimulate the processing and aggregation of amyloid-beta (Abeta), protect the protein from proteolysis, and increase the formation of neurofibrillary tangles by inducing the hyperphosphorylation of tau protein. These effects may be competitively inhibited by GAG. We have studied the effects of orally (by gavage) and subcutaneously (s.c.) administered low molecular weight heparin, C3 (4-10 oligosaccharides; MW = 2.1 kDa; USP value = 12 U/mg), on abnormal tau-2 protein immunoreactivity in the rat hippocampus following a single, unilateral intra-amygdaloid administration of Abeta(25-35). Oral administration of C3 (25 mg/kg; once daily) was initiated 3 days prior to Abeta(25-35) administration, and was continued daily for an additional 14 days. S.c. administration of C3 (2.5 mg/kg, twice daily), was started 3 days prior to, and was continued for 32 days after, Abeta(25-35) administration. Animal brains were subsequently processed for tau-2, ChAT-immunoreactivity, choline acetyltransferase (ChAT) activity and acetylcholinesterase (AChE) activity. Both oral and s.c. administration of C3 attenuated Abeta(25-35) induced appearance of tau-2-immunoreactive (IR) perikarya in the ipsilateral hippocampus (P < 0.05). Hippocampal cholinergic enzyme activity in C3 treated animals was not significantly different from control animals. The present findings suggest that C3 might be used successfully to prevent abnormal tau protein formation in chronic neurologic diseases, such as AD. Moreover, our data demonstrate that the mechanism of this effect does not appear to influence the cholinergic system of the brain.

Amyloid-beta injection in rat amygdala alters tau protein but not mRNA expression.

Previously we demonstrated local and distant changes in tau protein immunoreactivity reminiscent of those seen in Alzheimer's disease (AD) following a unilateral injection of amyloid-beta (Abeta)(25-35) into the rat amygdala. To explore the relevance of these findings to AD, we compared the effects of Abeta(1-42) to those of Abeta(25-35). Injections of both Abeta(1-42) and Abeta(25-35) into rat amygdala resulted in increased tau-2 immunolabeling in neurons. To determine whether these alterations were due to changes in the expression of tau, we measured tau protein expression by Western blotting and tau mRNA isoform expression by the reverse transcription-polymerase chain reaction in the amygdala, hippocampus, and cerebellum following a unilateral injection of Abeta(25-35) or vehicle into the amygdala. The levels of tau proteins were increased bilaterally in the amygdala of Abeta(25-35)- compared to vehicle-treated animals 8 and 16 days following treatment. The molecular weights of tau proteins were decreased in the Abeta(25-35)-treated (59-69 kDa) compared to the vehicle-treated (67-72 kDa) animals 8 days following treatment. There were no changes in tau mRNA expression in any brain region examined. In this model, just as in AD, there is an increase in tau protein levels without a change in tau mRNA expression, suggesting that Abeta peptides may influence tau protein stability in both the rat and the human brain.

Age and species-dependent differences in the neurokinin B system in rat and human brain.

Neurokinin B and its cognate neurokinin-3 receptor are expressed more in the forebrain than in brain stem structures but little is known about the primary function of this peptide system in the central processing of information. In general, few studies have specifically addressed age-related changes of tachykinins, notably the changes in number and/or distribution of the neurokinin B-expressing and neurokinin-3 receptor-bearing neurons. Data on functions and changes of neurokinins in physiological aging are limited and apply mainly to the substance P/neurokinin-1 receptor system. In the present study, we analyzed neurokinin B/neurokinin-3 receptor system in young (5 months) versus middle aged (15 months) and old rats (23-25 months) and also in aging human brains. For the majority of the immunohistochemically examined regions of the rat brain, there was no statistically significant change in neuronal number and size of the neurokinin B and neurokinin-3 receptor staining. In the adult human brain, there was no age-associated change of the number or size of neurokinin-B-positive neurons. However, we found a major decline in number of neurokinin-3 receptor-expressing neurons between young/middle aged (30 years to 69 years) versus old (70 years and older) adults. Interestingly, numbers of neurokinin-3 receptor-positive microglia increased whereas the neurokinin-3 receptor-positive astrocytes remained unchanged in both aging rat and human brains. Finally, in addition to assessing the morphological and quantitative changes of the neurokinin B/neurokinin-3 receptor system in the rat and human brain, we discuss functional implications of the observed interspecies differences.

Neurokinin-3 receptor distribution in rat and human brain: an immunohistochemical study.

Autoradiographic and immunohistochemical studies have shown that the neurokinin-3 receptor is widely distributed in the rodent CNS. Expression of the neurokinin-3 receptor in human brain, however, has been debated. These conflicting findings, as well as the poor resolution of autoradiographic images, prompted us to develop a polyclonal antibody against an oligopeptide derived from the carboxy-terminus consensus sequence of both the rat and human neurokinin-3 receptor ([C]ASTTSSFISSPYTSVDEYS, amino acids 434-452 of the rat neurokinin-3 receptor). Western blot analysis of both human and rat brain tissue revealed a major band in the molecular weight range 65,000-67,000, the proposed molecular weight of the neurokinin-3 receptor based on its amino acid sequence and presumed glycosylation state. The distribution of selective high affinity neurokinin-3 receptor agonist [3H]senktide binding and neurokinin-3 receptor immunoreactivity were virtually identical in the brains of male Fischer 344 rats. The highest concentrations of neurokinin-3 receptors were observed in cortical layers IV-V; the basolateral amygdaloid nucleus; the hypothalamic paraventricular, perifornical and supraoptic nuclei; the zona incerta; and the entopeduncular and interpeduncular nuclei. [3H]senktide binding and neurokinin-3 receptor immunoreactivity were compared in homologous cortical areas of the human and rat brain. In contrast to the rat, autoradiographic analysis of normal control human brains (35-75 years) revealed a distinct and predominant superficial cortical labeling in the glia limitans and the cortical layer I. However, neurokinin-3 receptor immunoreactivity could be found not only in the superficial cortical layers, but also on pyramidal neurons and astrocytes in the neuropil and white matter. These findings suggest species differences in both the cellular and anatomical distribution of the neurokinin-3 receptor.

Laterality in the histological effects of injections of amyloid-beta 25-35 into the amygdala of young Fischer rats.

We have observed that single amyloid-beta 25-35 (A beta) injections (5.0 nmol) into the right amygdala of rats produce progressive cytoskeletal and astrogliotic reactions not only within the amygdala, but also in distal brain regions that project to the amygdala. To determine if these effects are potentiated by bilateral injections, we injected A beta (5.0 nmol) into the left and right amygdala of young male Fischer rats. Animals were sacrificed 32 days postoperatively. Bilateral infusions of A beta induced significant neuronal shrinkage, tau-2 neuronal staining, and reactive astrocytosis within the right amygdala and/or hippocampus, compared with vehicle-treated rats. Surprisingly, the same brain regions within the left hemisphere were significantly less affected even though no differences were observed between the left and right amygdala in the size of Congored-positive A beta deposits. Unilateral injections of A beta into the left amygdala led to significant histological changes in the right amygdala and hippocampus, but not in the same brain regions within the left hemisphere. These results suggest a laterality in the histopathological effects of A beta in male Fischer rats. Identification of the cause for the lateralized effect of A beta may prove valuable for understanding the etiology of Alzheimer disease and provide possible therapeutic strategies designed to slow the progression of the disease.

Allograft prosthetic composite replacement for bone tumors.

Limb salvage for bone tumors has become the standard method of treatment. This technique involves removal of large segments of bone, most commonly around the hip and knee. Various types of reconstructive options are currently available, including osteoarticular allograft arthroplasty, modular oncology prosthetic arthroplasty, allograft prosthetic composite (APC) arthroplasty, and arthrodesis. Compared to other techniques, APC arthroplasty has many advantages, including restoration of bone stock, customization with conventional implant components, soft tissue attachment of tendons and ligaments, and preservation of the medullary canal of the host bone. The disadvantages of this technique include slow healing in the presence of chemotherapy, the possibility of disease transmission, and availability. The technique is suited either for aggressive benign tumors or for low-grade sarcomas where chemotherapy is not necessary. Further, it represents a good alternative for a failed modular oncology prosthesis, and also for the failed osteoarticular allograft because it restores bone stock. Good functional results have been reported with APC replacements, but long-term follow-up is needed to determine their durability.

Bilateral injections of amyloid-beta 25-35 into the amygdala of young Fischer rats: behavioral, neurochemical, and time dependent histopathological effects.

To examine the time course of the histopathological effects of bilateral injections of amyloid-beta 25-35 (A beta) and to determine if these effects are associated with a reduction in choline acetyltransferase activity and behavioral impairments, we injected A beta (5.0 nmol) into the amygdala of young male Fischer rats. Control rats received vehicle infusions. For histological analysis, animals were sacrificed at 8, 32, 64, 96, and 128 days postoperatively (n = 21-33 per timepoint). A beta induced neuronal tau-2 staining in the right, but not the left amygdala and hippocampus. A beta also induced reactive astrocytosis and neuronal shrinkage within the right hippocampus and amygdala, respectively. As with tau-2, these same brain regions within the left hemisphere in the A beta-treated rats were significantly less affected. In addition, A beta appeared to induce microglial and neuronal interleukin-1beta staining. The histopathological effects of A beta peaked at 32 days postoperatively but were not associated with a reduction in amygdaloid choline acetyltransferase activity. In a separate experiment, behavioral effects of bilateral intra-amygdaloid injections of A beta were analyzed at 34-52 days postoperatively. In an open field test, the treatment groups differed only in the numbers of rears emitted (p = 0.016). There was no effect of A beta in the Morris water maze or in the acquisition and retention of a one-way conditioned avoidance response. These data suggest a laterality in the histopathological effects of A beta and that the effects of single injections are in part transient. These findings also suggest a direct association between plaque and tangle formation in Alzheimer's disease, and support the use of this rat model to screen drugs that may alter the initial pathological events associated with Alzheimer's disease, that occur before the manifestations of extensive behavioral impairments become evident.

Local and distant histopathological effects of unilateral amyloid-beta 25-35 injections into the amygdala of young F344 rats.

To determine if amyloid-beta (A beta) induces tau-immunoreactivity (IR) and reactive astrocytosis in vivo, we injected A beta 25-35 (5.0 nmol) into the right amygdala of rats. At 8 days postinjection, the peptide induced tau-2 IR in neuronal cell bodies and processes ipsilaterally in the amygdala, cingulate cortex, and hippocampus. At 32 days postinjection, the intensity of tau-2 IR was greater than at 8 days in the amygdala and hippocampus, but not in the cingulate cortex. Induction of Alz-50 IR also was progressive but the morphology and distribution was different from tau-2 IR. Beaded fibers with occasional neuronal perikarya were visualized with Alz-50, and the IR was primarily observed in the ipsilateral amygdala. In addition, amygdaloid injections of A beta 25-35 induced reactive astrocytosis, particularly in the ipsilateral hippocampus at 32 days postoperatively. To our knowledge, this is the first study to show that in vivo injections of A beta 25-35 induce progressive transsynaptic cytoskeletal and astrogliotic reactions, that gradually spread from the area of injection to brain regions that have prominent efferent connections with that area. These findings also suggest a direct association between plaque and tangle formation in Alzheimer's disease.

beta-Amyloid 25-35 and/or quinolinic acid injections into the basal forebrain of young male Fischer-344 rats: behavioral, neurochemical and histological effects.

beta-Amyloid peptides have been shown to potentiate the neurotoxic effect of excitatory amino acids in vitro. In order to determine if this occurs in vivo, four experiments were performed. We injected beta-amyloid 25-35 (beta A 25-35) and/or quinolinic acid (QA) bilaterally into the ventral pallidum/substantia innominata (VP/SI) of rats. Control rats received vehicle infusions. A high dose of QA (75.0 nmol/3 microliters) increased open field activity and impaired spatial learning in the Morris water maze, but did not affect the acquisition of a one-way conditioned avoidance response. These changes were associated with histological evidence of neurotoxicity and a reduction in amygdaloid but not frontal cortical or hippocampal choline acetyltransferase (ChAT) activity. A lower dose of QA (37.5 nmol/3 microliters) produced no behavioral effects. It reduced amygdaloid ChAT activity to a lesser extent than the higher dose (15% vs. 29-37%), and caused less histological damage. beta A 25-35 (1.0 or 8.0 nmol/3 microliters) failed to produce behavioral, histological or neurochemical signs of toxicity. Neither dose of beta A 25-35 potentiated the effects of QA (37.5 nmol) on behavior or amygdaloid ChAT activity, and did not appear to increase the histological damage caused by QA. These results suggest that in vivo beta A 25-35 is not neurotoxic and does not potentiate the neurotoxicity of QA in the VP/SI. Further, the histological effects of a high dose of beta A 25-35 (8.0 nmol/3 microliters; a cavitation containing a Congo red positive proteinaceous material) are quite distinct from those produced by a high dose of QA (75.0 nmol/3 microliters; widespread neuronal loss and gliosis).