Geda Unabia

Transcriptional profiling of spinal cord injury‐induced central neuropathic pain

Central neuropathic pain (CNP) is an important problem following spinal cord injury (SCI), because it severely affects the quality of life of SCI patients. As in the patient population, the majority of rats develop significant allodynia (CNP rats) after moderate SCI. However, about 10% of SCI rats do not develop allodynia, or develop significantly less allodynia than CNP rats (non‐CNP rats). To identify transcriptional changes underlying CNP development after SCI, we used Affymetrix DNA microarrays and RNAs extracted from the spinal cords of CNP and non‐CNP rats. DNA microarry analysis showed significantly increased expression of a number of genes associated with inflammation and astrocytic activation in the spinal cords of rats that developed CNP. For example, mRNA levels of glial fibrilary acidic protein (GFAP) and Aquaporin 4 (AQP4) significantly increased in CNP rats. We also found that GFAP, S100β and AQP4 protein elevation persisted for at least 9 months throughout contused spinal cords, consistent with the chronic nature of CNP. Thus, we hypothesize that CNP development results, in part, from dysfunctional, chronically “over‐activated” astrocytes. Although, it has been shown that activated astrocytes are associated with peripheral neuropathic pain, this has not previously been demonstrated in CNP after SCI.

Acute and chronic changes in aquaporin 4 expression after spinal cord injury

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.

Application of the Avidin-Biotin-Peroxidase Complex (ABC) Method to the Light Microscopic Localization of Pituitary Hormones'

The avidin-biotin-peroxidase complex (ABC) method was applied to semithin (0.5-1 micron) plastic-embedded sections of intact male rat pituitaries with the use of techniques previously developed for the peroxidase-antiperoxidase complex (PAP) method. Stains for adrenocorticotropin (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were cleaner, more reliable, and more efficient. The ABC method allowed the use of the same high dilutions of primary antisera used with the PAP method. Incubation time was cut to a third of the time used for the PAP stain. Furthermore, if the incubation time matched that used with the PAP method, (24-48 hr), the antisera could be diluted 2- to 4-fold further. This enhanced specific staining and allowed the use of dilutions similar to those used in the radioimmunoassay. In agreement with Hsu and Raine (J Histochem Cytochem 29:1349, 1981), the ABC method produced staining after only a 1-4 hr incubation in primary antibody that was diluted optimally for the PAP complex method. The stain was weak, however, and cell counts showed that it was restricted to the fraction of the specific cell population which stored the most hormone. Our tests showed that the most convenient incubation times for optimal staining were 12-16 hr. Furthermore, the ABC method appeared to stabilize greatly the reaction for FSH and thus improved its precision and reliability.

Propentofylline attenuates allodynia, glial activation and modulates GABAergic tone after spinal cord injury in the rat.

&NA; In this study, we evaluated whether propentofylline, a methylxanthine derivative, modulates spinal glial activation and GABAergic inhibitory tone by modulation of glutamic acid decarboxylase (GAD)65, the GABA synthase enzyme, in the spinal dorsal horn following spinal cord injury (SCI). Sprague–Dawley rats (225–250 g) were given a unilateral spinal transverse injury, from dorsal to ventral, at the T13 spinal segment. Unilateral spinal injured rats developed robust bilateral hindlimb mechanical allodynia and hyperexcitability of spinal wide dynamic range (WDR) neurons in the lumbar enlargement (L4–L5) compared to sham controls, which was attenuated by intrathecal (i.t.) administration of GABA, dose‐dependently (0.01, 0.1, 0.5 μg). Western blotting and immunohistochemical data demonstrated that the expression level of GAD65 protein significantly decreased on both sides of the lumbar dorsal horn (L4/5) after SCI (p < 0.05). In addition, astrocytes and microglia showed soma hypertrophy as determined by increased soma area and increased GFAP and CD11b on both sides of the lumbar dorsal horn compared to sham controls, respectively (p < 0.05). Intrathecal treatment with propentofylline (PPF 10 mM) significantly attenuated the astrocytic and microglial soma hypertrophy and mechanical allodynia (p < 0.05). Additionally, the Western blotting and immunohistochemistry data demonstrated that i.t. treatment of PPF significantly prevented the decrease of GAD65 expression in both sides of the lumbar dorsal horn following SCI (p < 0.05). In conclusion, our present data demonstrate that propentofylline modulates glia activation and GABAergic inhibitory tone by modulation of GAD65 protein expression following spinal cord injury.

Exogenous Bcl-xl fusion protein spares neurons after spinal cord injury

Spinal cord injury (SCI) induces neuronal death, including apoptosis, which is completed within 24 hr at and around the impact site. We identified early proapoptotic transcriptional changes, including upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl‐xL, Bcl‐2, and Bcl‐w, using Affymetrix DNA microarrays. Because Bcl‐xL is the most robustly expressed antiapoptotic Bcl‐2 molecule in adult central nervous system, we decided to characterize better the effect of SCI on Bcl‐xL expression. We found Bcl‐xL expressed robustly throughout uninjured spinal cord in both neurons and glia cells. We also found Bcl‐xL localized in different cellular compartments: cytoplasmic, mitochondrial, and nuclear. Bcl‐xL protein levels decreased in the cytoplasm and mitochondria 2 hr after SCI and persisted for 24 hr. To test the contribution of proapoptotic decreases in Bcl‐xL to neuronal death, we augmented endogenous Bcl‐xL levels by administering Bcl‐xL fusion protein (Bcl‐xL FP) into injured spinal cords. Bcl‐xL FP significantly increased neuronal survival, suggesting that SCI‐induced changes in Bcl‐xL contribute considerably to neuronal death. Because Bcl‐xL FP increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically relevant in preserving sensory and motor functions after SCI.

Aquaporin 1 - a novel player in spinal cord injury.

The role of water channel aquaporin 1 (AQP‐1) in uninjured or injured spinal cords is unknown. AQP‐1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP‐1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant four‐ to eightfold increases in AQP‐1 levels at the site of injury (T10) persisting up to 11 months post‐contusion, a novel finding. Delayed AQP‐1 increases were also found in cervical and lumbar segments, suggesting the spreading of AQP‐1 changes over time after SCI. Given that the antioxidant melatonin significantly decreased SCI‐induced AQP‐1 increases and that hypoxia inducible factor‐1α was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP‐1 increases after SCI. Interestingly; AQP‐1 levels were not affected by long‐lasting hypertonicity that significantly increased astrocytic AQP‐4, suggesting that the primary role of AQP‐1 is not regulating isotonicity in spinal cords. Based on our results we propose possible novel roles for AQP‐1 in the injured spinal cords: (i) in neuronal and astrocytic swelling, as AQP‐1 was increased in all surviving neurons and reactive astrocytes after SCI and (ii) in the development of the neuropathic pain after SCI. We have shown that decreased AQP‐1 in melatonin‐treated SCI rats correlated with decreased AQP‐1 immunolabeling in the dorsal horns sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP‐1 and chronic neuropathic pain after SCI.

Nuclear factor‐κB decoy amelioration of spinal cord injury‐induced inflammation and behavior outcomes

Spinal cord injury (SCI) results in a pathophysiology characterized by multiple locomotor and sensory deficits, resulting in altered nociception and hyperalgesia. SCI triggers an early and prolonged inflammatory response, with increased interleukin‐1β levels. Transient changes are observed in subunit populations of the transcription factor nuclear factor‐κB (NF‐κB). There were decreases in neuronal c‐Rel levels and inverse increases in p65 and p50 levels. There were no changes in neuronal p52 or RelB subunits after SCI at any time point tested. Similarly, SCI had no effect on oligodendroglial levels of any NF‐κB subunit. There were significant early increases in COX‐2 and inducible nitric oxide synthase mRNA and protein levels after SCI. We used synthetic double‐stranded “decoy” deoxyoligonucleotides containing selective NF‐κB protein dimer binding consensus sequences. Decoys targeting the p65/p50 binding site on the COX‐2 promoter decreased SCI‐induced cell losses, NF‐κB p65/p50 DNA‐binding activity, and COX‐2 and iNOS protein levels. NF‐κB p65/p50 targeted decoys improved early locomotor recovery after moderate but not severe SCI, yet ameliorated SCI‐induced hypersensitization after both moderate and severe SCI. To determine whether changes in GABA activity played a role in decreased hypersensitivity after SCI and p65/p50 targeted decoy, we counted γ‐aminobutyric acid (GABA)‐containing neurons in laminae 1–3. There were significantly more GABAergic neurons in the p65/p50 targeted decoy‐treated group at the level of injury.

Application of a rapid avidin--biotin--peroxidase complex (ABC) technique to the localization of pituitary hormones at the electron microscopic level.

The new avidin--biotin--peroxidase complex (ABC) technique was applied to ultrathin sections of rat pituitary that were fixed with glutaraldehyde and embedded in Araldite 6005. The primary antisera dilutions that are normally applied for 24-48 hr with the peroxidase-antiperoxidase (PAP) complex technique were used. High background was observed with the ABC method when incubation times were 12-48 hr. Tests were then conducted with shorter incubation times. The staining intensity was measured with a densitometer. Detectable stain was seen after only 15 min in dilutions of 1:10,000 anti-bovine luteinizing hormone (bLH beta), 1:8000 anti-rat thyroid-stimulating hormone (rTSH beta), and 1:20,000 anti-25-39-adrenocorticotropic hormone (25-39ACTH). Optimal LH staining was seen after 30 min, whereas optimal staining for TSH or ACTH required 1 hr. Stain was detectable with a dilution of 1:4000 anti-human follicle-stimulating hormone (hFSH beta) after 30 min and was optimal after 4 hr. Prolonged incubation times with these dilutions decreased the staining intensity because a deposit of high background was produced that appeared as a filigreed network over the cells. When higher dilutions were tested with 2-hr incubation times, optimal staining was seen with 1:30,000 anti-bLH beta, 1:24,000 anti-rTSH beta, 1:30,000 anti-25-39ACTH, and 1:8000 anti-hFSH beta. These tests demonstrate the potential of the ABC method for the rapid detection of small amounts of specific and nonspecific antibodies that are bound to pituitary cells.

Cytochemical Studies of the Effects of Activin on Gonadotropin-releasing Hormone (GnRH) Binding by Pituitary Gonadotropes and Growth Hormone Cells

Activin stimulates the synthesis and secretion of follicle-stimulating hormone (FSH). It inhibits the synthesis and release of growth hormone (GH). It acts on gonadotropes by stimulating the synthesis of gonadotropin-releasing hormone (GnRH) receptors. To test activins effects on GnRH target cells, pituitary cells from diestrous or proestrous rats were exposed to media with and without 60 ng/ml activin for 24 hr and stimulated with biotinylated GnRH (Bio-GnRH). The populations were double-labeled for Bio-GnRH and/or luteinizing hormone-β (LH-β), FSH-β, or GH antigens. In both diestrous and proestrous rats, activin stimulated more LH and FSH cells and increased the percentages of GnRH target cells. In diestrous rats, activin stimulated increases in the average area and density of Bio-GnRH label on target cells. In addition, more FSH, LH, and GH cells bound Bio-GnRH. The increment in binding by gonadotropes was not as great as that normally seen from diestrus to proestrus, suggesting that additional factors (such as estradiol) may be needed. These data suggest that activin plays an important role in the augmentation of Bio-GnRH target cells normally seen before ovulation. Its actions on GH cells may reflect a role in the transitory change from a somatotrope to a somatogonadotrope that is seen from diestrus to proestrus.

Expression of follistatin mRNA by somatotropes and mammotropes early in the rat estrous cycle.

We previously found follistatin (FS) mRNA in gonadotropes [predominantly in cells with luteinizing hormone (LH) antigens] and folliculostellate cells (with S100 antigens) in diestrus rats pituitaries. However, earlier in the cycle, when percentages of gonadotropes are lowest, percentages of cells expressing FS are 1.5-2-fold higher than in diestrus. This study was designed to detect FS mRNA and other pituitary antigens to identify the additional cells with dual in situ hybridization and immunolabeling protocols. The mRNA was detected with biotinylated complementary oligonucleotide probes and avidin-biotin-peroxidase complexes. Significant labeling for FS mRNA was found in cells with the following antigens: growth hormone (GH) (7% of pituitary cells); prolactin (PRL) (5%); S100 protein (5%); follicle-stimulating hormone (FSH beta) (4%); LH beta (3%); and thyroid-stimulating hormone (TSH beta) (3%). Optimal conditions for detection included: overnight plating of > 50,000 cells/well (24-well tray) in media containing 10% fetal bovine serum; hybridization at 37 degrees C; and fixation in 2% glutaraldehyde. Whereas FS is expressed predominantly by LH gonadotropes at midcycle, FS mRNA can be expressed by all types of antigen-bearing cells earlier in the cycle. Its function in the pituitary may relate to its role in binding activin, which would result in inhibition of FSH release. However, since activin inhibits secretion of GH, PRL, and adrenocorticotropin (ACTH), FS may also control activins effects on these cells. The FS-expressing cells may therefore be paracrine or autocrine regulators.