Hamster Brain Tissue Research Articles

Two-Dimensional Correlation Spectroscopy for Multimodal Analysis of FT-IR, Raman, and MALDI-TOF MS Hyperspectral Images with Hamster Brain Tissue.

Hyperspectral imaging (HSI) techniques are useful for obtaining very detailed structural and compositional information from biomedical, pharmaceutical, or clinical samples, among others. The informative value of these methods can be further increased through the application of different HSI techniques and joint analysis of the data. However, interpretation and understanding of multimodal HSI have been impeded by difficulties in registration of the different HSI data sets and by the lack of integrative analysis concepts. Here, we introduce two-dimensional correlation spectroscopy (2DCOS) as a novel technique for jointly analyzing HSI data which allows one to obtain deeper insights into the chemistry of complex samples by decrypting auto- and heterospectral correlations that may exist between features of the different HSI data. The general workflow of 2DCOS analysis is demonstrated by HSI examples acquired from cryo-sections of hamster brain tissue using Fourier-transform infrared (FT-IR) microspectroscopy, confocal Raman microspectroscopy (CRM), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Multimodal hyperspectral image analysis by 2DCOS opens up new opportunities for spectral band assignments and thus the interpretation of structure-spectra and composition-spectra relationships. We foresee wide application potential for describing complex samples in various fields ranging from biomedicine to industrial applications.

Human variant Creutzfeldt-Jakob disease and sheep scrapie PrPres detection using seeded conversion of recombinant prion protein

The pathological isoform of the prion protein (PrP(res)) can serve as a marker for prion diseases, but more practical tests are needed for preclinical diagnosis and sensitive detection of many prion infections. Previously we showed that the quaking-induced conversion (QuIC) assay can detect sub-femtogram levels of PrP(res) in scrapie-infected hamster brain tissue and distinguish cerebral spinal fluid (CSF) samples from normal and scrapie-infected hamsters. We now report the adaptation of the QuIC reaction to prion diseases of medical and agricultural interest: human variant Creutzfeldt-Jakob disease (vCJD) and sheep scrapie. PrP(res)-positive and -negative brain homogenates from humans and sheep were discriminated within 1-2 days with a sensitivity of 10-100 fg PrP(res). More importantly, in as little as 22 h we were able to distinguish CSF samples from scrapie-infected and uninfected sheep. These results suggest the presence of prions in CSF from scrapie-infected sheep. This new method enables the relatively rapid and sensitive detection of human CJD and sheep scrapie PrP(res) and may facilitate the development of practical preclinical diagnostic and high-throughput interference tests.

Establishment of a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters infected with scrapie agent 263K

To establish a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters and to address the stability of the panel conserved under the specific condition, for evaluating the diagnostic techniques of human and animal's prion diseases. 30 brain tissues of hamsters infected with scrapie strain 263K intracerebrally and 30 ones of normal hamsters were enrolled in this panel. Each brain sample was prepared to 10%, 1% and 0.5% homogenates and aliquoted into stocks. The presences of PrP(Sc) in each brain sample were evaluated with PrP-specific Western Blots and partially with immunohistochemistry, and the stability of PrP(Sc) signals in each sample were repeatedly assessed half a year later and 3 years later. PrP(Sc) signals were detected in all stocks of 10% brain homogenates from infected hamsters, 26 out of 30 stocks of 1% homogenates and 19 out of 30 stocks of 0.5% homogenates. The assessments of PrP(Sc) signals in all samples half-year and three years later demonstrated almost unchanged. All homogenates of brain tissues of normal hamsters were PrP(Sc) negative. A prion disease PrP(Sc) panel of the brain tissues, which includes 90 PrP(Sc) positive stocks and PrP(Sc) negative ones, was successfully established, with a reliable stability of PrP(Sc) signals.

Analyses of the expressions of GFAP in the brain tissues of hamsters infected with various amounts of scrapie strain 263K at terminal stage

To investigate whether gliosis in the brain tissues of the hamsters infected with various amounts of scrapie strain 263K is correlated with the inoculation doses or the incubation times. The total values of glial fibrillary acidic protein (GFAP) in brains were evaluated by Western Blots and the GFAP-stained cells were detected by immunohistochemistry (IHC). The characteristics of GFAP distributions among various groups were defined by quantitive and statistic analyses. Compared with the brain tissues of normal hamsters, remarkably higher total GFAP levels and more GFAP-stained cells were observed in the brain tissues of infected ones, howbeit, no significant difference was addressed among the infected groups. Inoculations of various amounts of scrapie strain 263K into experimental hamsters intracerebrally induced the similar patterns of gliosis in the brains at the clinically terminal stage, regardless of infectious doses and incubation times.

Different expression patterns of CK2 subunits in the brains of experimental animals and patients with transmissible spongiform encephalopathies

To address the possible alteration of casein kinase 2 (CK2) in transmissible spongiform encephalopathies (TSEs), the levels and patterns of CK2 in the brain tissues of hamsters or C57BL mice inoculated intracerebrally with scrapie agents 263K or 139A were evaluated by Western blots, followed by quantitative analysis. Specific semi-quantitative RT-PCR for evaluating the mRNA transcripts of CK2 subunits was performed in parallel. Compared with normal animals, the levels of CK2alpha and CK2beta in the brains of infected hamsters and mice were significantly decreased, regardless of which scrapie agent was. However, the expression of CK2alpha' or CK2alpha'/CK2alpha'' in the animals infected with agents 263K or 139A was considerably increased. Furthermore, decreases of CK2alpha and CK2beta and increases of CK2alpha'/CK2alpha'' were observed in cerebella homogenates from one familial Creutzfeldt-Jakob disease (fCJD) case and one fatal familial insomnia (FFI) case. These results suggest that alterations of CK2 subunits in brains are illness-correlative phenomena in TSEs and indicate a potential linkage of CK2 changes with the pathogenesis of prion diseases.

Structural characterization of β-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro

The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrP C) into a misfolded isoform (PrP Sc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90–231). A novel in vitro conversion assay based on aerobic incubation of PrP in the presence of elemental copper pellets at pH 5 was established, resulting in aggregation of highly β-sheeted prion proteins. We show for the first time that two discrete oligomeric species of elongated shape, approx. 25 mers and 100 mers, are formed on the pathway of oxidative PrP aggregation in vitro, which are well characterized regarding shape and size using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and electron microscopy (EM). Considering that small oligomers of highly similar size have recently been reported to show the highest specific infectivity within TSE-infected brain tissues of hamsters, the novel oligomers observed in this study are interesting candidates as agent causing neurodegenerative and/or self-propagating effects. Moreover, our results significantly strengthen the theory that oxidative stress might be an influence that leads to substantial structural conversions of PrP in vivo.

Expression of PrP C as HIS-fusion form in a baculovirus system and conversion of expressed PrP-sen to PrP-res in a cell-free system

Conversion of the PrP cellular form (PrP C) to the pathogenic form (PrP Sc) is the key step in the pathogenesis of transmissible spongiform encephalophathies. Although the mechanism of conformational conversion of PrP proteins remains uncertain, the cell-free conversion reaction and other in vitro PrP amplification tests allow it to be studied under the much quicker and simpler conditions than those of transmission bioassay in vivo. Using baculovirus expression system, wild-type hamster (HaPrP) and human PrP (HuPrP), as well as D178N mutated human PrP (HuPrPm178) were expressed in HIS-fusion form. After 35 S-methionine labeling and purification with Ni–NTA agarose affinity chromatography, individual expressed PrP proteins were mixed with PrP Sc isolated from hamster brain tissue infected with scrapie 263K. Protease-resistant isoform was detected in the homologous HaPrP reaction, but not in the two heterologous HuPrP preparations, implying a species-specific molecular recognition between PrP C and PrP Sc. HIS-tag in HIS–HaPrP seems to have little effect on the formation of protease-resistant protein in this preparation. This system proposes a simple and protein productive-enriched way for cell-free conversion of prion proteins, as the replacement of native or genetic engineering expressed sole PrP C from mammalian or non-mammalian sources.

Neuronal Apoptosis Mediated by IL-1β Expression in Viral Encephalitis Caused by a Neuroadapted Strain of the Mumps Virus (Kilham Strain) in Hamsters

The neuroadapted Kilham strain of the mumps virus produces lethal encephalitis in newborn hamsters after intracerebral inoculation. The pathogenesis of this encephalitis is not fully understood, but recently, apoptosis and associated cytokine production have been recognized to be major pathologic mechanisms by which viruses cause injury to neuronal host cells. To analyze the main factors producing brain injury in this viral encephalitis, the following questions were investigated: (1) does the virus induce neuronal apoptosis and (2) does expression of cytokines regulate the induction of neuronal apoptosis? Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was used as a marker of neuronal apoptosis and TUNEL-positive neurons were widespread in the infected cerebral cortex. DNA fragmentation yielding DNA ladders characteristic of apoptosis was also observed in infected hamster brain tissue. Apoptotic cells in infected brains were observed after the appearance of inflammatory changes. Overexpression of IL-1β, but not TNF-α or Fas-L, was clearly detected in infected brains, as determined by Western blot and RT-PCR. Immunohistochemistry revealed a striking correlation between IL-1β expression and neuronal apoptosis. Injection of recombinant IL-1β into normal hamster brain resulted in neuronal apoptosis in cerebral cortex. On the other hand, neutralizing IL-1β antibodies decreased the number of cells undergoing apoptosis in infected hamster brains and subsequent death. We conclude that the fatal encephalitis induced by the Kilham strain of the mumps virus is mediated by immunopathological processes and that overexpression of IL-1β, which mediates the induction of neuronal apoptosis, may play a major role in these processes.

Inhibition of collagenolytic activity relates to quantitative reduction of invasion in vitro in a c-Ha-ras transfected glial cell line.

The function of proteases in brain tumor invasion is currently not well established. For tumors of epithelial and fibromatous origin collagenase production can enhance the invasive capacity of cells to penetrate basement membranes. We showed previously that a c-Ha-ras transformed glial cell line (CxT24neo3) invaded hamster brain tissue in vivo. These cells were also capable of invading reconstituted basement membrane and embryonic chick hearts in vitro. Since the histopathology of CxT24neo3 tumors mimics that of glioblastoma multiforme in humans, CxT24neo3 was used as the model in vitro for this type of brain tumor. Presently, we detected by zymogram analysis a gelatinase that was secreted by CxT24neo3 and that had an apparent molecular mass of 62 kD. To verify whether gelatinase affected invasion in vitro of these glial cells we determined the efficacy of a substrate specific collagenase inhibitor on invasion in vitro. GM6001 is a synthetic polypeptide that specifically occupies the substrate binding sites of metalloprotease. Since this drug did not show cytotoxicity, its specificity for metalloprotease is a valuable tool to evaluate the physiological function of these enzymes on invasion. We found that treatment of CxT24neo3 with GM6001 reduced the fraction of invading CxT24neo3 cells through reconstituted basement membrane. These data suggest that metalloproteases can stimulate brain tumor invasion.

In vitro translation in a hamster brain cell-free system

We have developed a cell-free translation system derived from hamster brain tissue. The optimal incorporation conditions were 160 mM K +, 2 mM Mg 2+ and 50 μM spermine. The absence of this latter compound could be compensated only by doubling (to 4 mM) Mg 2+ concentration. This system was inhibited by cycloheximide and emetine (elongation inhibitors) as well as NaF and aurintricar☐ilic acid (ATA) (initiation inhibitors). The nature of the ATA inhibiting effect was studied by kinetic comparison with cycloheximide and polyribosome sedimentation pattern.

Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.

The colocalization of substance P and prodynorphin immunoreactivity in neurons of the medial preoptic area, bed nucleus of the stria terminalis and medial nucleus of the amygdala of the Syrian hamster

To determine the extent of colocalization of substance P (SP) and prodynorphin peptides within neurons of the medial nucleus of the amygdala (AMe), medial bed nucleus of the stria terminalis (BNSTm) and medial preoptic area (MPOA), we incubated colchicine-treated Syrian hamster brain tissue in an antiserum mixture containing rat anti-SP antibody combined with 1 of 3 rabbit antibodies against prodynorphin peptides: anti-dynorphin A(1–17), anti-dynorphin B(1–13) or anti-C-peptide. This was followed by incubation in a secondary antiserum mixture containing fluorescein-labelled anti-rabbit and rhodamine-labelled anti-rat antibodies. Sections were viewed with an epifluorescence microscope using blue light excitation for fluorescein and green light excitation for rhodamine. Colocalization of SP and prodynorphin labelling was observed in neurons of the caudal parts of AMe, BNSTm and MPOA, areas which are essential for male mating behavior. The colocalization was most extensive in the dorsolateral part of the caudal MPOA, the caudodorsal part of the BNSTm, and in the posterodorsal subdivision of AMe. Although all 3 dynorphin peptides coexisted with SP in these areas, dynorphin B did so less than C-peptide, and dynorphin A less than dynorphin B.

Isolation of measles virus polypeptides from infected brain tissue by affinity chromatography

A method has been developed to isolate measles virus proteins from infected hamster brain tissue. Suckling hamsters inoculated intracerebrally with the HBS strain of measles virus were used in these studies. Viral proteins were isolated from infected brain lysates by affinity chromatography on Sepharose beads coupled with IgG from rabbit hyperimmune anti-measles serum. The eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretically transferred onto blotting matrix, and immunolabelled with anti-measles antibodies. Individual viral proteins were identified by labelling with monoclonal or monospecific antibodies. All viral proteins except the fusion (F 1) protein were identifiable on the immunoblots in relative amounts comparabe to purified virions. In addition, a second phosphoprotein (P) band not found in purified virions was present in infected brains and cell cultures infected with HBS or LEC strains of virus. This method should be useful for isolating small quantities of viral proteins from large amounts of tissue, and should make possible the characterization of measles virus proteins in persistently infected CNS tissue.

Immunoglobulin G from subacute sclerosing panencephalitis brain as an immunological reagent

Proteins were extracted at pH 2.5 from carefully washed brain homogenates of patients with subacute sclerosing panencephalitis. Immunoglobulin G was isolated from the extracts by gel filtration and immunoadsorbent chromatography and was 125I radioiodinated. The labeled immunoglobulin G was reacted with purified measles virus antigens by crossed immunoelectrophoresis and utilized to identify antigens in measles-infected cell cultures and hamster brain tissue subjected to treatment corresponding to that used for elution of immunoglobulin G. The method described is useful in identifying the specific nature of antibodies as well as antigens in tissue-associated immune complexes.

Induction of Hamster T Cell-Specific Antiserum in Rabbits Using Hamster Brain Tissue

Antiserum specific for hamster thymus-derived lymphocytes, prepared by immunizing rabbits with hamster brain tissue, was cytotoxic to hamster thymocytes greater than lymph node cells greater than spleen cells, while virtually non-reactive against bone marrow. This antiserum inhibited spleen cell response to the T cell mitogen, phytohemagglutinin, but not to lipopolysaccharide, a B cell mitogen.

Viability and infectivity of measles virus-infected nervous tissue frozen in cryoprotective medium.

Measles virus-infected hamster brain tissue remained viable when frozen slowly in medium containing dimethyl sulfoxide. This procedure was essential for isolation of neurotropic measles virus from tissue specimens stored frozen.

Viability and Infectivity of Measles Virus-Infected Nervous Tissue Frozen in Cryoprotective Medium

Measles virus-infected hamster brain tissue remained viable when frozen slowly in medium containing dimethyl sulfoxide. This procedure was essential for isolation of neurotropic measles virus from tissue specimens stored frozen.

General characteristics of the cells transformed in vitro by human adenovirus type 12.

From in vivo induced tumors by adenovirus type 12, four cell lines were established in vitro (H-1, H2-1, H2-2 and H-4) and from the in vitro transformed foci in embryonal hamster brain tissue culture by the same virus, 7 lines were successfully subcultivated (HT-1, HT-2 so on). There was no gross differences in their characteristics. Morphologically they were tenaciously adherent small epithelioid cells. They needed large inoculum to subculture in rubber stoppwred culture system. Cellular aggregations were frequently observed in the static culture conditions and calcium concentration suggested to bear it. The attempt to isolate the infectious virus from tumor cells and transformed cells at various passage levels has failed so far.