Histochemical study of the pre—and postnatal development of acetylcholinesterase in the rat spinal cord

The distribution of acetylcholinesterase(AChE)-positive structures in the developing rat spinal cord was studied with AChE-histochemistry.AChE-positive perikarya were first seen on embryonic day 14(E14) in the ventrolateral portion of the spinal cord.From that time onward.AChE=containing cells appeared gradually in the intermediate gray,dorsal horn and lateral spinal nucleus of the spinal cord in a ventral-to-dorsal,and lateral-to-medial order.No obvious rostral-to-caudal sequence was found.At birth,the distribution pattern of AChE-positive perikarya was basically similar to that in adults.After birth a dramatic increase in the AChE staining intensity extended from postnatal day 5(P5) to postnatal day 21(P21),In addition,two phases of transient AChE staining were observed in the external surface of the dorsal horn from embryonic day 15(E15) to embryonic day 21(E21) and in the marginal layer from embryonic day 21(E21) to postnatal day 14(P14),respectively.     

Oral administration of melatonin modulates the expression of tumor necrosis factor-α (TNF-α) gene in irradiated rat cervical spinal cord

AimWe aimed to determine the changes in TNF-α expression and Malondialdehyde (MDA) level in a short time after irradiation. Furthermore, we evaluated the effect of melatonin on the modulation of TNF-α gene expression.BackgroundThe radio-sensitivity of the cervical spinal cord limits the dose of radiation which can be delivered to tumors in the neck region. There is increasing evidence that TNF-α has a role in the development of the acute phase of spinal cord injury.Materials/MethodsFour groups of rats were investigated. Group 1 (vehicle treatment) served as the control. Group 2 (radiation) was treated with the vehicle, and 30 min later, the rats were exposed to radiation. Group 3 (radiation + melatonin) was given an oral administration of melatonin (100 mg/kg body weight) and 30 min later exposed to radiation in the same manner as in group 2. Group 4 (melatonin-only) was also given an oral administration of melatonin (100 mg/kg body weight). 5 mg/kg of melatonin was administered daily to rats in groups 3 and 4, and the vehicle was administered daily to rats in groups 1 and 2.ResultsThree weeks after irradiation, TNF-α gene up-regulated almost 5 fold in the irradiated group compared to the normal group. TNF-α gene expression in the melatonin pretreatment group, compared to the radiation group, was significantly down-regulated 3 weeks after irradiation (p < 0.05). MDA levels increased after irradiation and then significantly decreased under melatonin treatment.ConclusionWe suggest that inhibition of TNF-α expression by oral administration of melatonin may be a therapeutic option for preventing radiation-induced spinal cord injury.     

Effect of busulphan treatment and elevated temperature on the expression of the β-pol gene in rat testis

Changes in the expression pattern of the DNA polymerase gene during inhibition of spermatogenesis by busulphan and by temperature (artificial cryptorchidism) have been studied. Transient arrest of spermatogenesis in two-month-old rats after injection of a single dose of busulphan (10 mg/kg) resulted in parallel but transient decrease in the 1.4 kb of -pol mRNA level to an undetectable value, followed by its reappearance after resumption of spermatogenesis. An artificial cryptorchidism also caused a drastic decrease of -pol mRNA level. Both results as well as morphological examination of testis after busulphan injection and artificial cryptochidism revealed that spermatocytes and spermatids represent the testicular cell fraction containing the elevated amount of -pol mRNA. Involvement of DNA polymerase in meiotic recombination is discussed.     

Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

To characterize the long-term effects of adolescent marijuana abuse, we performed a proteomic analysis of cerebellar extracts from adult female rats with and without ovariectomy that were treated with Δ9-THC for 40 days during adolescence. Six proteins were found to significantly differ among the four treatment groups, with Δ9-THC and ovariectomy (OVX) decreasing the mitochondrial proteins, pyruvate carboxylase and NADH dehydrogenase, whereas the levels of putative cytosolic molecular chaperones NM23B, translationally controlled tumor protein, DJ-1 and activator of heat-shock 90kDa protein ATPase homolog 1 (AHA1) were increased. We further analyzed the effects of AHA1, a HSP90 co-chaperone, on CB1R and CB2R trafficking and signaling in transfected HEK293T and Neuro-2A cells. In HEK293T cells, AHA1 over-expression enhanced plasma membrane levels of CB1R and increased CB1R-mediated effects on cAMP levels and on MAPK phosphorylation. AHA1 over-expression also enhanced cell surface levels of endogenous CB1R and the effects of Δ9-THC on the cAMP levels in Neuro-2A cells. In contrast, over-expression of AHA1 did not affect the subcellular localization and signaling of CB2R. Our data indicate that chronic Δ9-THC administration in adolescence altered the endogenous levels of specialized proteins in the cerebellum, such as AHA1, and that this protein can change CB1R cell surface levels and signaling.     

Colocalization of protein kinase C ⊺-subtype and calcitonin gene-related peptide in rat spinal cord

Summary Colocalization of calcitonin gene-related peptide (CGRP) and protein kinase C -subtype (PKC-) like immunoreactivities (LI) and the subcellular localization of CGRP-LI were studied in the ventral horn of rat spinal cord. Ultrastructurally CGRP-LI was localized on the membranes of the Golgi-complexes, in multivesicular bodies and in vesicles adjacent to the Golgi-complex in motoneuron perikarya. The colocalization of PKC- and CGRP-LI was detected in most of the ventral horn motoneurons. However, few motoneurons were only PKC--immunoreactive. These results suggest that PKC- may be involved in the regulation of CGRP release from motoric axon terminals.     

Co-culture of bovine muscle satellite cells with preadipocytes increases PPARγ and C/EBPβ gene expression in differentiated myoblasts and increases GPR43 gene expression in adipocytes

We hypothesized that preadipocyte differentiation would be depressed by differentiating myoblasts, whereas preadipocytes would promote adipogenic gene expression in myoblasts in a co-culture system. We also determined the effects of arginine, a biological precursor of nitric oxide, and/or trans-10, cis-12 conjugated linoleic acid (CLA) on adipogenic gene expression during differentiation of bovine preadipocytes and myoblasts. Bovine semimembranosus satellite cells (BSC) and subcutaneous preadipocytes were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's modified Eagle medium (DMEM) and 1% antibiotics during the 3-day proliferation period. After proliferation, BSC and preadipocytes were treated for 3 days with 3% horse serum/DMEM and 5% FBS/DMEM with antibiotics, respectively. Media also contained 100 μM oleic acid, 10 μg/ml insulin, 1 μg/ml pioglitazone and 1 μg/ml dexamethasone. Subsequently, the differentiating myoblasts and adipocytes were cultured in their respective media with 5 mM arginine and/or 40 μM trans-10, cis-12 CLA for 4 days. Finally, myoblasts and adipocytes were single- or co-cultured for 2 h singly or in combination. Arginine stimulated SCD gene expression, whereas CLA depressed SCD gene expression in adipocytes and myoblasts (P=.002). Co-culture of adipocytes and myoblasts elicited an increase in C/EBPβ and PPARγ gene expression in differentiated myoblasts (P≤.01) and an increase in GPR43 gene expression in adipocytes (P=.01). Expression of AMPKα and CPT1ß was unaffected by co-culture, although SCD gene expression tended (P=.12) to be depressed by co-culture. These experiments demonstrated that co-culture of adipocytes with myoblasts increased adipogenic gene expression in the myoblastic cells.     

Cystathionine β-synthase in structural elements of the human brain and spinal cord

By means of the immunohistochemical method, the presence and distribution of cystathionine β-synthase (CBS) was studied in nerve cells of the spinal cord and brainstem nuclei in eight men aged 18–44 years who had died as a result of causes not connected with damage to the central nervous system. CBS-positive neurons are revealed in all studied brain parts, in which their content varied in different nuclei from 0.9 to 17%. Large cells of motor nuclei more often had high and very high density of the reaction product deposition. In sensory nuclei the high portion was of small neurons with low intensity of the enzymatic reaction.     

Δ-Tetrahydrocannabinol regulates the p53 post-translational modifiers Murine double minute 2 and the Small Ubiquitin MOdifier protein in the rat brain

The phytocannabinoid Δ⁹-Tetrahydrocannabinol (Δ⁹-THC), the main psychoactive cannabinoid in cannabis, activates a number of signalling cascades including p53. This study examines the role of Δ⁹-THC in regulating the p53 post-translational modifier proteins, Murine double minute (Mdm2) and Small Ubquitin-like MOdifier protein 1 (SUMO-1) in cortical neurons. Δ⁹-THC increased both Mdm2 and SUMO-1 protein expression and induced the deSUMOylation of p53 in a cannabinoid receptor type 1 (CB₁)-receptor dependent manner. We demonstrate that Δ⁹-THC decreased the SUMOylation of the CB₁ receptor. The data reveal a novel role for cannabinoid receptor activation in modulating the SUMO regulatory system.

Structured summary

MINT-7266621: Cb1 (uniprotkb:P20272) physically interacts (MI:0915) with SUMO-1 (uniprotkb:Q5I0H3) by anti bait coimmunoprecipitation(MI:0006)MINT-7266633: SUMO-1 (uniprotkb:Q5I0H3) and Cb1 (uniprotkb:P20272) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7266611: p53 (uniprotkb:P10361) physically interacts (MI:0915) with SUMO-1 (uniprotkb:Q5I0H3) by anti bait coimmunoprecipitation (MI:0006)     

Up-regulation of Μ-opioid receptors in the spinal cord of morphine-tolerant rats

Though morphine remains the most powerful drug for treating pain, its effectiveness is limited by the development of tolerance and dependence. The mechanism underlying development of tolerance to morphine is still poorly understood. One of the factors could be an alteration in the number of Μ-receptors within specific parts of the nervous system. However, reports on changes in the Μ-opioid receptor density in the spinal cord after chronic morphine administration are conflicting. Most of the studies have used subcutaneously implanted morphine pellets to produce tolerance. However, it does not simulate clinical conditions, where it is more common to administer morphine at intervals, either by injections or orally. In the present study, rats were made tolerant to morphine by injecting increasing doses of morphine (10-50 mg/kg, subcutaneously) for five days.In vitro tissue autoradiography for localization of Μ-receptor in the spinal cord was done using [³H]-DAMGO. As compared to the spinal cord of control rats, the spinal cord of tolerant rats showed an 18.8% increase or up-regulation in the density of Μ-receptors in the superficial layers of the dorsal horn. This up-regulation of Μ-receptors after morphine tolerance suggests that a fraction of the receptors have been rendered desensitized, which in turn could lead to tolerance     

Δ9-tetrahydrocannabinol and dimethylheptylpyran induced tachycardia in the conscious rat

Since cannabinoids lead to dose-related tachycardia in man but dose dependent bradycardia has been reported thus far in laboratory animals, there would seem to be a need for an experimental model in which the effect seen in man (tachycardia) could be reproduced and explored. In the conscious rat, the compounds Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and dimethylheptylpyran (DMHP) injected i.p. led to dose-related increases in heart rate at 10–20 minutes after administration. In vehicle (ethanol) control rats there were small increases in heart rate. Propranolol given before Δ⁹-THC resulted in a parallel shift to the right of the dose-effect curve. Adrenalectomy led to a significant (p<0.01) decrease in tachycardia following Δ⁹-THC and DMHP while ganglionic block markedly decreased the heart rate increases after Δ⁹-THC (p<0.001). Systolic blood pressure at nearly all doses of Δ⁹-THC was minimally affected, although it tended to decrease with increasing dose. Tachycardia in the rat may be the result of a centrally mediated release of epinephrine from the adrenal gland.     

Silencing CD36 gene expression results in the inhibition of latent-TGF-β1 activation and suppression of silica-induced lung fibrosis in the rat

Background

The biologically active form of transforming growth factor-β1 (TGF-β1) plays a key role in the development of lung fibrosis. CD36 is involved in the transformation of latent TGF-β1 (L-TGF-β1) to active TGF-β1. To clarify the role of CD36 in the development of silica-induced lung fibrosis, a rat silicosis model was used to observe both the inhibition of L-TGF-β1 activation and the antifibrotic effect obtained by lentiviral vector silencing of CD36 expression.

Methods

The rat silicosis model was induced by intratracheal injection of 10 mg silica per rat and CD36 expression was silenced by administration of a lentiviral vector (Lv-shCD36). The inhibition of L-TGF-β1 activation was examined using a CCL-64 mink lung epithelial growth inhibition assay, while determination of hydroxyproline content along with pathological and immunohistochemical examinations were used for observation of the inhibition of silica-induced lung fibrosis.

Results

The lentiviral vector (Lv-shCD36) silenced expression of CD36 in alveolar macrophages (AMs) obtained from bronchoalveolar lavage fluid (BALF) and the activation of L-TGF-β1 in the BALF was inhibited by Lv-shCD36. The hydroxyproline content of silica+Lv-shCD36 treated groups was significantly lower than in other experimental groups. The degree of fibrosis in the silica+Lv-shCD36-treated groups was less than observed in other experimental groups. The expression of collagen I and III in the silica+Lv-shCD36-treated group was significantly lower than in the other experimental groups.

Conclusion

These results indicate that silencing expression of CD36 can result in the inhibition of L-TGF-β1 activation in a rat silicosis model, thus further preventing the development of silica-induced lung fibrosis.     

Abnormal myelination in the spinal cord of PTPα-knockout mice

PTPα interacts with F3/contactin to form a membrane-spanning co-receptor complex to transduce extracellular signals to Fyn tyrosine kinase. As both F3 and Fyn regulate myelination, we investigated a role for PTPα in this process. Here, we report that both oligodendrocytes and neurons express PTPα that evenly distributes along myelinated axons of the spinal cord. The ablation of PTPα in vivo leads to early formation of transverse bands that are mainly constituted by F3 and Caspr along the axoglial interface. Notably, PTPα deficiency facilitates abnormal myelination and pronouncedly increases the number of non-landed oligodendrocyte loops at shortened paranodes in the spinal cord. Small axons, which are normally less myelinated, have thick myelin sheaths in the spinal cord of PTPα-null animals. Thus, PTPα may be involved in the formation of axoglial junctions and ensheathment in small axons during myelination of the spinal cord.     

“Axonal spheroids” in the spinal cord of normal rabbits

Summary Within the gray matter and the white matter of the spinal cord of apparently healthy rabbits, myelinated and unmyelinated axonal swellings, so called axonal spheroids, occur. Most of the spheroids contain mitochondria, dense bodies, vesicles and fragments of the tubular or smooth endoplasmic reticulum. In myelinated spheroids the process of swelling is effected by slippage of the myelin leaflets. At the periphery of the unmyelinated parts of the spheroids, synapses are regularly found. The presynaptic terminal bouton is formed by the spheroid. A few myelinated and unmyelinated spheroids are packed with fine granular material while mitochondria are lacking. The axonal spheroids may represent a physiological, perhaps age dependent phenomenon.Dedicated to Prof. Dr. Berta Scharrer on the occasion of her 70^th birthdayThe author wishes to thank Mrs. Helga Zuther-Witzsch, Mrs. Elisabeth Schöngarth and Miss Hildegard Schöning for excellent technical assistance. Supported by the Deutsche Forschungsgemeinschaft, Projekt Le 69/7-13     

Quantifying the internal deformation of the rodent spinal cord during acute spinal cord injury – the validation of a method

Visualization and analysis of the rodent spinal cord subject to experimental spinal cord injury (SCI) has almost completely been limited to naked-eye observations, and a single measure of gross spinal cord motion due to injury. This study introduces a novel method which utilizes MRI to quantify the deformation of the rodent spinal cord due to imposed, clinically-relevant injuries – specifically, cervical contusion and dislocation mechanisms. The image registration methods were developed using the Advanced Normalization Tools package, which incorporate rigid, affine and deformable registration steps. The proposed method is validated against a fiducial-based, ‘gold-standard’ measure of spinal cord tissue motion. The validation analysis yielded accuracy (and precision) values of 62 μm (49 μm), 73 μm (79 μm) and 112 μm (110 μm), for the medio-lateral, dorso-ventral and cranio-caudal directions, respectively. The internal morphological change of the spinal cord has never before been quantified, experimentally. This study demonstrates the capability of this method and its potential for future application to in vivo rodent models of SCI.