Microwave-accelerated bioassay technique for rapid and quantitative detection of biological and environmental samples.

Quantitative detection of molecules of interest from biological and environmental samples in a rapid manner, particularly with a relevant concentration range, is imperative to the timely assessment of human diseases and environmental issues. In this work, we employed the microwave-accelerated bioassay (MAB) technique, which is based on the combined use of circular bioassay platforms and microwave heating, for rapid and quantitative detection of Glial Fibrillary Acidic Protein (GFAP) and Shiga like toxin (STX 1). The proof-of-principle use of the MAB technique with the circular bioassay platforms for the rapid detection of GFAP in buffer based on colorimetric and fluorescence readouts was demonstrated with a 900W kitchen microwave. We also employed the MAB technique with a new microwave system (called the iCrystal system) for the detection of GFAP from mice with brain injuries and STX 1 from a city water stream. Control bioassays included the commercially available gold standard bioassay kits run at room temperature. Our results show that the lower limit of detection (LLOD) of the colorimetric and fluorescence based bioassays for GFAP was decreased by ~1000 times using the MAB technique and our circular bioassay platforms as compared to the commercially available bioassay kits. The overall bioassay time for GFAP and STX 1 was reduced from 4h using commercially available bioassay kits to 10min using the MAB technique.

Proteomic profiling of the retinas in a neonatal rat model of oxygen-induced retinopathy with a reproducible ion-current-based MS1 approach.

Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.

A DNA-based nano-immunoassay for the label-free detection of glial fibrillary acidic protein in multicell lysates.

We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cell's differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper. FROM THE CLINICAL EDITOR: This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP.

Identification and characterization of citrulline-modified brain proteins by combining HCD and CID fragmentation.

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high-resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys-C, and Glu-C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher-energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID-triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.

Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products.

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.

Comparison of analytical methods for the detection of central nervous system tissue in ground beef.

Bovine spongiform encephalopathy is most likely transmitted by the consumption of central nervous system tissue of infected cows. The objective of this study was to compare the sensitivity or limits of detection of two central nervous system tissue detection assays (glial fibrillary acidic protein enzyme-linked immunosorbent assay [ELISA] and neuron-specific enolase Western blotting assay) in by-products and ground beef. By-products including brain, spinal cord, and ileum were collected from the slaughterhouse and used for analyses with ELISA and Western blotting assays. Beef samples were prepared by mixing ground beef with different amounts of spinal cord tissue (0, 0.03, 0.06, and 0.1%) and were analyzed using the two central nervous system tissue detection methods. Both analytical assays were applicable in detecting central nervous system tissue in ground beef. However, the ELISA method was considered superior because of its ease of use, high sensitivity, and rapidity as compared with the Western blot method.

Alternative cutting methods to minimize transfer of nervous system tissue during steak preparation from bone-in short loins.

Fresh beef products, such as steaks, may become contaminated with potential specified risk materials (SRMs), such as central nervous system tissue, during the fabrication of bone-in loin subprimals. The objective of this study was to evaluate current and alternative cutting methods that could be used to minimize the transfer of nervous system tissue (NST) tissue during preparation of steaks from bone-in short loins. Bone-in short loins were cut according to three methods. (i) Cutting method I-The vertebral column bones were removed prior to cutting the loin into steaks from the medial (vertebral column) to lateral (flank) side. (ii) Cutting method II--The loin was cut into steaks from the vertebral column side to the flank side prior to removal of the vertebral column bones. (iii) Cutting method III--The loin was cut into steaks from the flank side to the vertebral column side prior to removal of the vertebral column bones. Results indicated that surface areas along the vertebral column cutting line had detectable (0.10 and 0.22% NST/100 cm2) and, thus, higher potential SRM contamination than resulting steak surfaces or the cutting blade. Overall, there were no detectable (<0.10% NST/100 cm2) differences in NST contamination of steaks produced by the three cutting methods. Immunohistochemical evaluation of areas on excised and ground steak surfaces indicated that regardless of cutting method, there was generally "no" to "moderate" staining, suggesting that detectable (0.137 to 0.201% NST) contamination from these samples was most likely due to peripheral nerve detection. These results imply that steaks may be cut from bone-in short loins prior to removal of the vertebral column bones without affecting the transfer of NST to resulting steaks at concentrations <0.10% NST/100 cm2.

Comparison of immunochemical (enzyme-linked immunosorbent assay) and immunohistochemical methods for the detection of central nervous system tissue in meat products.

Three methods are widely used in the United States to detect the presence of central nervous system (CNS) tissue in meat products: the fluorescent enzyme-linked immunosorbent assay (F-ELISA), developed in this laboratory, the colorimetric Ridascreen Risk Material 10/5 ELISA (R-ELISA), and the U.S. Department of Agriculture, Food Safety and Inspection Service immunohistochemical (IHC) procedure. These assays are based on the immunological detection of glial fibrillary acidic protein (GFAP), a neural antigen largely restricted to the CNS. The objective of the current study was to compare the sensitivity and repeatability of these tests for detecting the presence of neural tissue in meat. Ground beef spiked with 0.05 to 0.5% of bovine brain, spinal cord (SC), or dorsal root ganglia, as well as advanced meat recovery samples, were evaluated by each of the three GFAP detection procedures. Interassay coefficients of variation for the F-ELISA GFAP standards were 7 to 25%, and intra-assay variation due to sampling and extraction of spiked ground beef was 7 to 13% for SC and 8 to 14% for brain (n = 10). The F-ELISA was the most sensitive of the methods tested, capable of detecting 0.3 ng GFAP standard per well and the presence of 0.05% brain and SC in meat. The R-ELISA standards produced highly variable results (up to 36% variation) and, as a result, none of these standards were different from zero (n = 26). The R-ELISA resulted in high sample variation in SC-spiked ground beef samples (coefficients of variation were 23 to 50%) and did not detect the presence of brain contamination. After modification of the R-ELISA sampling and extraction methods, SC-spiked sample variation was reduced to 16 to 20%, and sensitivity was improved from 0.3 to 0.2% SC, although brain tissue was still not detected. The IHC analysis of CNS-adulterated ground beef had a sensitivity of 0.2% SC and 0.05% brain, with false-negative rates of 10 to 20% at and above the stated sensitivities. None of the methods examined detected dorsal root ganglia contamination. The F-ELISA detected the presence of CNS contamination in 20% of the advanced meat recovery samples, compared to 3.5 to 5% for the R-ELISA and 2% for IHC. This study suggests that the F-ELISA is much more sensitive and repeatable than either the R-ELISA or the IHC procedure method for the detection of CNS tissue in meat products.

Hot boning of intact carcasses: a procedure to avoid central nervous system self-contamination in beef and beef products.

Compared with traditional boning of split refrigerated carcasses, hot boning of intact carcasses (the removal of meat from the skeleton prerigor) provides several commercially important cuts, may improve quality and reduce refrigeration costs, and may reduce the contamination of carcasses with central nervous system (CNS) tissue. In a comparative study of hot boning of intact and split carcasses, the CNS tissue contamination of intact carcasses was negligible (as measured with the CNS-related proteins glial fibrillary acidic protein and S-100 protein), but split carcasses were highly contaminated. The same trends were observed for dissection worktables used during the boning process. Most current boning plants have processing lines that are organized for boning carcass quarters, where the carcasses, in addition to transversal division, also are split horizontally. This part of the boning process was incorporated in the design of our study. Nine of the 18 intact carcasses were split horizontally between thoracic vertebrae 10 and 11 before they were hot boned. CNS tissue contamination was not detected on the carcass site related to this procedure. The amount of CNS tissue contamination was similar in boned cuts and minced meat from split and intact carcasses, except in the forerib. Boning of split carcasses appears to reduce CNS tissue contamination significantly to a level comparable to that of intact hot-boned carcasses.

Identification of nitrated proteins in the normal rat brain using a proteomics approach.

BACKGROUND: The nitration of tyrosine has been suggested to play a role in the pathogenesis of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). METHODS: In the present study, we identified four targets of protein nitration, T-complex polypeptide 1 alpha subunit (TCP-1), neurofilament L (NFL), glial fibrillary acidic protein (GFAP) and clathrin heavy chain (CHC), in the normal rat cortex using a proteomics approach. CONCLUSIONS: There have been no reports on these proteins being identified by proteomics as nitrated forms in the brain. For further study, we have to investigate alterations in these nitrated proteins during aging and in neurodegenerative disorders.

Proteomic analysis of glial fibrillary acidic protein in Alzheimer's disease and aging brain.

Chronic inflammation is known to play an important role in the heterogeneous pathogenesis of Alzheimer's disease (AD). Activated astrocytes expressing glial fibrillary acidic protein (GFAP) are closely associated with AD pathology, such as tangles, neuritic plaques and amyloid depositions. Altogether, 46 soluble isoforms of GFAP were separated and most of them quantified by two-dimensional immunoblotting in frontal cortices of AD patients and age-matched controls. A 60% increase in the amount of more acidic isoforms of GFAP was observed in AD and these isoforms were both phosphorylated and N-glycosylated, while more basic isoforms were O-glycosylated and exhibited no quantitative differences between post-mortem AD and control brains. These data highlight the importance of exploring isoform-specific levels of proteins in pathophysiological conditions since modifications of proteins determine their activity state, localization, turnover and interaction with other molecules. Mechanisms, structures and functional consequences of modification of GFAP isoforms remain to be clarified.

In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of rats.

Procedures for the preparation of high- and low-salt Triton insoluble cytoskeletal fractions from rat brain suitable for studying in vitro phosphorylation by endogenous kinases and phosphatases are described. The high-salt Triton insoluble cytoskeletal fraction is enriched in neurofilament subunits (NF-H, NF-M and NF-L), vimentin and glial fibrillary acidic protein (GFAP), while the low-salt Triton insoluble cytoskeletal fraction contains detergent insoluble cytoskeletal elements such as intermediate filament subunits and tubulins. One of our approaches is to incubate cerebral cortex slices with [32P]orthophosphate before the cytoskeletal fraction extraction, which allows the in vitro phosphorylation of cytoskeletal constituents in an intact intracellular environment. On the other hand, we also incubate low- or high-salt cytoskeletal fractions previously prepared with [gamma(32)P]ATP. By doing so, we are able to study the direct effects of substances on the kinase and phosphatase activities associated with the cytoskeletal fraction. Moreover by using specific activators or inhibitors of protein kinases and phosphatases we can obtain more detailed information on the alterations provoked by these substances. These approaches are useful for the investigation of the neurotoxic effects of various drugs and metabolites affecting the cytoskeletal-associated phosphorylation system in the brain.

Reverse transcription-polymerase chain reaction assay for species-specific detection of bovine central nervous system tissue in meat and meat products.

This paper reports the development of a reverse transcription-polymerase chain reaction (RT-PCR) assay coupled with restriction fragment length polymorphism (RFLP) analysis to specifically detect the glial fibrillary acidic protein (GFAP) mRNA of bovine central nervous system (CNS) tissue in minced meat and meat products. RNA extracted from bovine brain tissue and brain tissue from other mammals yielded a 168-bp CNS-specific signal after RT-PCR. The species specificity of the assay can be obtained by subsequent RFLP analysis of the amplified RT-PCR product. To determine the tissue specificity of the RT-PCR assay, various bovine tissues were analyzed. Bovine GFAP mRNA was detected in the brain and spinal cord, and these results are consistent with the reported large amounts of detectable protein in these tissues. Additionally, GFAP mRNA was present in skeletal muscle tissue samples and in some heart muscle tissue samples, but heat treatment of samples prior to extraction resulted in the loss of the non-CNS signals. To evaluate the stability and detectability of bovine GFAP mRNA in comminuted meat and in cooked meat products, mixtures containing bovine brain homogenate at concentrations of 0.5 to 5% were prepared and analyzed. The examination of minced meat with added bovine brain homogenate revealedGFAP mRNA RT-PCR signal stability for at least 7 days at a storage temperature of 4 degrees C. In cooked meat products bovine GFAP mRNA signal was detectable for at least 35 days. Bovine brain homogenate at a concentration of 0.5% was successfully detected in all of the experiments conducted, and no false-negative results were obtained. It is concluded that bovine GFAP mRNA can serve as a sensitive and specific marker for bovine CNS tissue in minced meat and in pasteurized meat products.

¿Es el fibrosarcoma primitivo cerebralla fase final de la evolucióndel gliosarcoma? / Is primitive brain fibrosarcoma the end stage in the evolution of gliosarcoma?

El gliosarcoma es un tumor cerebral poco frecuente que viene definido por una proliferación bifásica en la que toman parte: a) una fase gliomatosa, habitualmente un glioblastoma, con expresión de GFAP y negatividad para la Vimentina; b) una fase conjuntiva con expresión de Vimentina y negatividad a la GFAP. Por su parte, el fibrosarcoma cerebral primitivo es un tumor muy infrecuente, que se define por una proliferación de células fusiformes, capaces de expresar Vimentina, incapaces de expresar GFAP y que producen abundantes fibras reticulares y, ocasionalmente, colágenas. En nuestro medio hemos estudiado dos casos de gliosarcoma por medio del cultivo de tejidos y por medio del cultivo orgánico. En ambos casos, el cultivo de tejidos demostró la población dual, en forma de colonias celulares diferentes, aun dentro del mismo cultivo. Por su parte, el cultivo orgánico demostró el crecimiento doble en las primeras fases del crecimiento, en tanto que el cabo del tiempo, la fase conjuntiva va predominando sobre la glial, hasta que esta última desaparece. En consecuencia, se plantea la posibilidad de que el fibrosarcoma primitivo cerebral represente la fase final de un gliosarcoma en el que el componente conjuntivo ha hecho desaparecer el componente glial inicial (AU)

Inervación de la pars interarticularis en la espondilolistesis / Innervation of the pars interarticularis on spondylolisthesis

En relación con la espondilolistesis ístmica lítica se ha referido que existen terminaciones nerviosas en dicho defecto fibroso, que se estimulan con los micromovimientos, originando dolor, que podría ser tratado con la reparación directa de la pars, a falta de otras posibles fuentes de síntomas, sin sacrificar el movimiento de ninguna unidad funcional vertebral. Es posible que con la edad, además de aparecer otras fuentes de dolor (como el disco intervertebral en su proceso degenerativo), disminuya el número de tales terminaciones nerviosas (a semejanza de algunas pseudoartrosis) o que, cuanto menos, varíe la presencia relativa de cada tipo de terminación, en un hipotético proceso de adaptación progresiva de una pseudoartrosis más o menos estable mecánicamente (la espondilólisis). Objetivo. Estudiar la variación con la edad de las terminaciones nerviosas en el defecto de la pars, en número y distribución relativa de los diversos tipos de terminaciones presentes, en pacientes con una espondilolistesis ístmica lítica con dolor de espalda. Material y método. Se tomaron biopsias bilaterales del defecto de la pars en 15 pacientes consecutivos tratados quirúrgicamente por espondilolistesis ístmica lítica. Las muestras se estudiaron con microscopia óptica con tinción de hematoxilina-eosina e inmunohistoquímica con técnicas S-100 y para proteína glial ácida y neurofilamentos. Resultados. Se encontraron sólo terminaciones nerviosas libres, en las biopsias de 8 de los 15 pacientes, sin constatar ninguna variación atribuible a la edad. Conclusión. La presencia de terminaciones nerviosas en el defecto fibroso de la pars interarticularis podría no ser un hallazgo general (AU)

Changes in the immunologic phenotype of human malignant glioma cells after passaging in vitro.

Although immunotherapeutic strategies against glioblastomas have been promising both in vitro and in animal models, similar successes have not been realized in human clinical trials. One reason may be that immunotherapeutic strategies are based on prior studies that primarily have used human glioblastoma cell lines passaged in vitro, which may not accurately reflect the in vivo properties of glioblastoma cells. In this report, we used flow cytometry to quantify the expression of immunological cell surface molecules on human glioblastomas directly ex vivo (prior to any in vitro culturing) and after varying passages in vitro. Furthermore, we used ELISA to quantitate cytokine secretion after various passages in vitro. We demonstrate that in vitro culturing of established cell lines led to increases in the cell surface expression of MHC class I and ICAM-1 and secretion of IL-6 and TGF-beta(2). Furthermore, there were significant changes in the expression of MHC class I, MHC class II, B7-2, ICAM-1, and FasL when comparing ex vivo tumor cells to those after a single passage in vitro. After passaging once in vitro, there were also significant changes in the secretion of TGF-beta(2) and IL-10. This report indicates that in vitro culturing leads to significant changes in both cell surface molecules and secreted cytokines, which are known to affect the ability of immune cells to initiate an anti-tumor immune response. These changes in the immunological phenotype of glioblastomas after in vitro culturing may in part explain the limited success of immunotherapeutic strategies against glioblastomas in human clinical trials.

The detection of central nervous system tissue on beef carcasses and in comminuted beef.

We report the development and validation of a fluorescent enzyme-linked immunosorbent assay (ELISA) for glial fibrillary acidic protein (GFAP), which can be used as a rapid and sensitive method to detect CNS tissue in meat products. The fluorometric assay is sensitive to 0.2 ng GFAP and has an intra-assay coefficient of variation (CV) of 2.0% and an interassay CV of 14.1%. Bovine spinal cord and brain demonstrate dose-response curves that are parallel to GFAP standards, whereas peripheral sciatic nerve and cervical ganglia also cross-react at high tissue levels. The use of another central nervous system marker, syntaxin 1-B, was not effective for neural tissue detection. Less than 1.0 ng GFAP per mg tissue was found on most beef subprimals and advanced meat recovery (AMR) product. Occasional samples contained higher levels of GFAP, probably because of contamination by the carcass-splitting saw, incomplete removal of the spinal cord, or a chance sampling of a major nerve. Further reduction of CNS content was facilitated by removal of the cervical vertebrae and the spinal canal prior to processing beef chuck bones through AMR equipment. The presence of GFAP was very low (0.037 ng/mg) in beef patties collected from major processors throughout the USA. The presence of normal sausage ingredients or heating the product to 80 degrees C for 60 min did not affect the detection of GFAP. Heating the product to 115 degrees C for 100 min eliminated the detectability of GFAP.

GDNF increases the density of cells containing calbindin but not of cells containing calretinin in cultured rat and human fetal nigral tissue.

Among the dopaminergic neurons in substantia nigra pars compacta and in the ventral tegmental area, subpopulations express the calcium-binding proteins calbindin (CB) and calretinin (CR), and the CB-containing neurons are supposed to be less prone to degeneration in Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic neurons. Using free-floating roller-tube (FFRT) cultures derived from fetal rat (E14) ventral mesencephalon we found that GDNF (10 ng/ml) significantly increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons. The possible effects of GDNF treatment on CB-immunoreactive (CB-ir) and CR-ir neurons in such cultures were examined in the present study. The neuronal cell densities were measured by quantifying the numbers of CB-ir and CR-ir neurons in areas of sections through the most extensive parts of the spherical cultures. In 4-day-old and 8-day-old cultures GDNF treatment increased the density of CB-ir neurons by 50% and 59%, respectively. Partial co-existence of TH and CB was shown using the method of double immunolabeling. The density of CR-containing neurons was unaffected by GDNF treatment as confirmed by Western blotting for CR. Parallel effects of GDNF treatment were obtained for cultures of human fetal ventral mesencephalon (8 weeks postconception). In conclusion, our findings identify GDNF as a potent factor for fetal rat and human nigral CB-ir neurons able to promote their survival in culture. Referring to a suggested neuroprotective role of CB, the results may be of relevance in the context of neuronal transplantation of patients suffering from severe Parkinson's disease.

Biocompatibility of poly (DL-lactide-co-glycolide) microspheres implanted into the brain.

The delivery of therapeutic molecules to the brain has been limited in part due to the presence of the blood-brain barrier. One potential solution is the implantation of biodegradable polymers with sustained release of drugs. Poly (DL-lactide-co-glycolide) (PLG) is a bioerodible polymer with a long and successful history of use as a suture material. More recently, PLG has been investigated for localized and sustained delivery of molecules into both peripheral sites and the brain. Despite its well-defined safety profile for parenteral applications, little information exists concerning the safety of PLG when implanted into the brain. To further characterize the biocompatibility of PLG in the brain, we examined the gliotic response following implants of PLG into the brains of rats. As a control, each animal received an injection of the suspension medium into the contralateral hemisphere. Following implantation, PLG was well tolerated. GFAP-positive astrocytes were observed throughout the cerebral cortex and striatum on both the implanted and control sides, with the reaction being greatest within the heavily myelinated fiber tracts of the corpus callosum. Quantitative analyses revealed that this reaction occurred within 1 h postsurgery, reached its peak at 1 week following surgery, and then decreased markedly by 1 month postsurgery. A minimal gliotic reaction was still present 1 year postsurgery but was localized to the needle tract. No differences in GFAP reactivity were seen between the polymer-implanted and control sides at any time point. Histological analysis determined that the majority of the PLG disappeared between 1 and 4 weeks. A set of parallel studies in which PLG samples were retrieved from the brain at various time points corroborated these findings and determined that the majority of PLG degraded within 2 weeks following implantation. Together, these results demonstrate that PLG is well tolerated following implantation into the CNS and that the astrocytic response to PLG is largely a consequence of the mechanical trauma that occurs during surgery. The biocompatibility of PLG implanted into the CNS provides further support for its use in a wide range of new therapeutic applications for sustained and localized drug delivery to the brain.

Expression of the Huntington's disease gene is regulated in astrocytes in the arcuate nucleus of the hypothalamus of postpartum rats.

Huntington's disease (HD) is one of a number of neurodegenerative disorders caused by expansion of polyglutamine-encoding CAG repeats within specific genes. Huntingtin, the protein product of the HD gene, is widely expressed in neural and nonneural human and rodent tissue. The function of the wild-type or mutated form of huntingtin is currently unknown. We have observed that relative to naive and male animals, huntingtin protein was significantly increased in the arcuate nucleus of postpartum rats. Using an oligonucleotide probe, in situ and Northern blot hybridization confirmed the expression of huntingtin mRNA. Quantification of the in situ hybridization signal in the arcuate nucleus revealed an approximate sevenfold increase in the expression of huntingtin mRNA in postpartum, lactating animals compared with naive female or male animals. Emulsion autoradiography and immunohistochemistry revealed that the cells with elevated huntingtin expression had a stellate conformation that morphologically resembled astrocytes. Dual label immunofluorescence immunohistochemistry demonstrated the colocalization of huntingtin and glial fibrillary acidic protein in these cells, confirming that they were astrocytes. Astrocytes expressing huntingtin were consistently found in close apposition to neuronal soma, suggesting interactions between these cell types. During the perinatal and postnatal period, the hypothalamus undergoes alterations in metabolic function. Our results support the idea of glia-induced metabolic changes in the hypothalamus. These results provide the first demonstration of naturally occurring changes in the expression of the Huntington's disease gene in the brain and suggest that huntingtin may play an important role in the processes that regulate neuroendocrine function.