Immunohistochemical Study of Immunophilin 1–15 Fragment in Intact Frog Brain,and in the Brain and Spinal Cord of Intact and Spinal Cord Hemisectioned Rats

Previously by immunohistochemical technique the distribution of immunophilin 1–15 fragment (IphF) isolated from bovine hypothalamus was examined in various tissues (heart, lung), including immune system organs (spleen and thymus) of intact rats. IphF-like immunoreactivity (IphF-LI) was revealed in several cell types: lymphocytes, monocytes, macrophages and mast cells. In the present study the immunohistochemical localization of IphF was examined in intact rat and frog brains. In rat brain several cell groups concentrated particularly in the supraoptic nucleus (SON) of hypothalamus, medulla oblongata (reticular formation, olives, hypoglossal and facial motor nuclei) and cerebellum (lateral cerebellar nucleus) demonstrated IphF-LI. In frog hypothalamus (SON) the same working dilution (1:5000) of IphF-antiserum revealed very strong immunoreactivity. In the paraventricular nucleus (PVN) IphF-LI varicosities were scattered around the immunonegative cells. The second cell groups showing IphF-LI in the frog brain were gliocytes (mainly the astrocytes). Besides, IphF distribution was investigated in rats subjected to hemisection of spinal cord (SC) with and without administration of proline-rich polypeptide (PRP). PRP was isolated from bovine neurohypophysis neurosecretory granules, produced by magnocellular nuclei of hypothalamus. Hemisection of SC led to changes of IphF distribution in the hypothalamus. In PRP treated animals IphF showed no immunoreactivity. PRP is suggested to act as a neurotransmitter and neuroregulator.     

Proteomic Analysis of PKCγ-Related Proteins in the Spinal Cord of Morphine-Tolerant Rats

Background

Morphine tolerance is a common drawback of chronic morphine exposure, hindering use of this drug. Studies have shown that PKCã may play a key role in the development of morphine tolerance, although the mechanisms are not fully known.

Methodology/Principal Findings

In a rat model of morphine tolerance, PKCã knockdown in the spinal cord was successfully carried out using RNA interference (RNAi) with lentiviral vector-mediated short hairpin RNA of PKCã (LV-shPKCã). Spinal cords (L4-L5) were obtained surgically from morphine-tolerant (MT) rats with and without PKCã knockdown, for comparative proteomic analysis. Total proteins from the spinal cords (L4-L5) were extracted and separated using two-dimensional gel electrophoresis (2DGE); 2D gel images were analyzed with PDQuest software. Seven differential gel-spots were observed with increased spot volume, and 18 spots observed with decreased spot volume. Among these, 13 differentially expressed proteins (DEPs) were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), comparing between MT rats with and without PKCã knockdown. The DEPs identified have roles in the cytoskeleton, as neurotrophic factors, in oxidative stress, in ion metabolism, in cell signaling, and as chaperones. Three DEPs (GFAP, FSCN and GDNF) were validated with Western blot analysis, confirming the DEP data. Furthermore, using immunohistochemical analysis, we reveal for the first time that FSCN is involved in the development of morphine tolerance.

Conclusions/Significance

These data cast light on the proteins associated with the PKCã activity during morphine tolerance, and hence may contribute to clarification of the mechanisms by which PKCã influences MT.     

Regional Changes of Cyclic 3′,5′-Guanosine Monophosphate in the Spinal Cord of the Rabbit following Brief Repeated Ischemic Insults

The regional distribution of cyclic 3,5-guanosine monophosphate was studied in the lumbosacral segments of the spinal cord of the rabbit under physiological conditions and following brief repeated sublethal ischemic insults. While the basal cGMP level in the gray matter was about 0.120 nmol cGMP/mg wet. wt., the level of cGMP in non-compartmentalized white matter was about half of this value. The highest level of cGMP in the compartmentalized gray matter was found in the dorsal horns, about 0.180 nmol cGMP/mg wet. wt., whereas the level of cGMP was greatly reduced in the ventral horns, reaching one half of the previous value. Multiple sublethal ischemic insults, repeated at 1-h intervals, caused a statistically significant decrease of cGMP in all gray matter regions. While the post-ischemic and post-reperfusion level of cGMP in the dorsal horns remained relatively high in comparison with the intermediate zone and ventral horns, the changes of cGMP level detected in the white matter columns differed considerably and resulted in a statistically significant cGMP increase in the dorsal and ventral columns and, vice versa, a statistically significant decrease of cGMP was found in the lateral columns.     

Transient Spinal Cord Ischemia in Rat: The Time Course of Spinal FOS Protein Expression and the Effect of Intraischemic Hypothermia (27°C)

1. In the present study, we characterize the time course of spinal FOS protein expression after transient noninjurious (6-min) or injurious (12-min) spinal ischemia induced by inflation of a balloon catheter placed into the descending thoracic aorta. In addition, this work examined the effects of spinal hypothermia on FOS expression induced either by ischemia or by potassium-evoked depolarization (intrathecal KCl).2. Short-lasting (6-min) spinal ischemia evoked a transient FOS protein expression. The peak expression was seen 2 hr after reperfusion in all laminar levels in lumbosacral segments. At 4 hr of reperfusion, more selective FOS expression in spinal interneurons localized in the central part of laminae V–VII was seen. At 24 hr no significant increase in FOS protein was detected.3. After 12 min of ischemia and 2 hr of reflow, nonspecific FOS expression was seen in both white and gray matter, predominantly in nonneuronal elements. Intrathecal KCl-induced FOS expression in spinal neurons in the dorsal horn and in the intermediate zone. Spinal hypothermia (27°C) significantly suppressed FOS expression after 6 or 12 min of ischemia but not after KCl-evoked depolarization.4. Data from the present study show that an injurious (but not noninjurious) interval of spinal ischemia evokes spinal FOS protein expression in glial cells 2 hr after reflow. The lack of neuronal FOS expression corresponds with extensive neuronal degeneration seen in this region 24 hr after reflow. Noninjurious (6-min) ischemia induced a transient, but typically neuronal FOS expression. The significant blocking effect of hypothermia (27°C) on the FOS induction after ischemia but not after potassium-evoked depolarization also suggests that simple neuronal depolarization is a key trigger in FOS induction.     

Diabetes-Related Alteration of Occludin Expression in Rat Blood–Spinal Cord Barrier

Occludin is an essential component of tight junctions, which are involved in controlling the integrity of the blood–brain barrier and blood–spinal cord barrier (BSCB). Diabetes-induced alteration of occludin in rat BSCB and the relationship between occludin level and disease course was examined. Diabetes was induced using streptozotocin. Occludin rat spinal cord mRNA levels were assessed by real-time quantitative RT-PCR. Protein levels were examined by western blot. Occludin expression in 1-month diabetic rats was significantly reduced compared to the controls (0.20 ± 0.01 vs 1.00 ± 0.01, respectively; P < 0.05). Expression was also significantly lower in the 3-month diabetic group (0.06 ± 0.02; P < 0.01). Occludin protein levels of 1-month (0.53 ± 0.01) and 3-month (0.31 ± 0.01) diabetic rats were also significantly reduced compared to controls (0.91 ± 0.06; P < 0.01 for both). Diabetes decreased BSCB occludin expression at the mRNA and protein level. This down-regulation appears to correlate with the course of the disease, and may be a causal factor of diabetes-induced increase of BSCB permeability.     

Early Changes of β-Catenins and Menins in Spinal Cord Dorsal Horn after Peripheral Nerve Injury

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Aβ-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. β-Catenin and menin as synaptogenic factors are critically involved in synapse formation. However, the roles of β-catenin and menin in neuropathic pain are still unclear. Using Western blot analysis we investigated the changes of β-catenin and menin in the spinal dorsal horn after unilateral spared nerve injury (SNI). We demonstrated an increase in both β-catenin and menin protein levels in the ipsilateral spinal dorsal horn at days 1 and 3 following spared nerve injury (P < 0.05). These increases were associated with changes in paw withdrawal threshold to mechanical stimuli and weight bearing deficit suggestive of pain behavior and spontaneous ongoing pain respectively. However, the injury-associated increases in β-catenins and menins levels returned to control levels at day 14. In conclusion, these results indicate that peripheral nerve injury induces upregulation of β-catenins and menins in the dorsal horn of the spinal cord, which may contribute to the development of chronic neuropathic pain. Antagonists of these molecules may serve as new therapeutic agents.     

Effects of Chronic Restraint Stress and 17-β-Estradiol Replacement on Oxidative Stress in the Spinal Cord of Ovariectomized Female Rats

Previous studies have shown sex-specific oxidative changes in spinal cord of rats submitted to chronic stress, which may be due to gonadal hormones. Here, we assessed total radical-trapping potential (TRAP), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and lipid peroxidation (evaluated by the TBARS test) in the spinal cord of ovariectomized (OVX) female rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided into controls and chronically stressed (for 40 days). Our findings demonstrate that chronic stress decreased TRAP, and increased SOD activity in spinal cord homogenates from ovariectomized female rats and had no effect on GPx activity. On the other hand, groups receiving 17β-estradiol replacement presented a decreased GPx activity, but no alteration in TRAP and in SOD activity. No differences in the TBARS test were found in any of the groups analyzed. In conclusion, our results support the idea that chronic stress induces an imbalance between SOD and GPx activities, additionally decreasing TRAP. Estradiol replacement did not reverse the effects of chronic stress, but induced a decrease in GPx activity. Therefore, estradiol replacement in ovariectomized chronically stressed rats could make the spinal cord more susceptible to oxidative injury.     

FK506 Attenuates the Inflammation in Rat Spinal Cord Injury by Inhibiting the Activation of NF-κB in Microglia Cells

FK-506 (Tacrolimus) is a very commonly used immunomodulatory agent that plays important roles in modulating the calcium-dependent phosphoserine–phosphothreonine protein phosphatase calcineurin and thus inhibits calcineurin-mediated secondary neuronal damage. The biological function of FK-506 in the spinal cord has not been fully elucidated. To clarify the anti-inflammatory action of FK-506 in spinal cord injury (SCI), we performed an acute spinal cord contusion injury model in adult rats and hypoxia-treated primary spinal cord microglia cultures. This work studied the activation of NF-κB and proinflammatory cytokine (TNF-a, IL-1b, and IL-6) expression. ELISA and q-PCR analysis revealed that TNF-a, IL-1b, and IL-6 levels significantly increased 3 days after spinal cord contusion and decreased after 14 days, accompanied by the increased activation of NF-κB. This increase was reversed by an FK-506 treatment. Double immunofluorescence labeling suggested that NF-κB activation was especially prominent in microglia. Immunohistochemistry confirmed no alteration in the number of microglia. Moreover, the results in hypoxia-treated primary spinal cord microglia confirmed the effect of FK-506 on TNF-a, IL-1b, and IL-6 expression and NF-κB activation. These findings suggest that FK-506 may be involved in microglial activation after SCI.     

Direct Effect of Remifentanil and Glycine Contained in Ultiva? on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses

Background

Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord.

Methods

We made patch-clamp recordings from substantia gelatinosa (SG) neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli.

Results

Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs) evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs) were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine.

Conclusions

We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal and intravenous administrations.     

Are the 10 Meter and 6 Minute Walk Tests Redundant in Patients with Spinal Cord Injury?

Objective

To evaluate the relationship and redundancy between gait speeds measured by the 10 Meter Walk Test (10MWT) and 6 Minute Walk Test (6MWT) after motor incomplete spinal cord injury (iSCI). To identify gait speed thresholds supporting functional ambulation as measured with the Spinal Cord Injury Functional Ambulation Inventory (SCI-FAI).

Design

Prospective observational cohort.

Setting

Seven outpatient rehabilitation centers from the Christopher and Dana Reeve Foundation NeuroRecovery Network (NRN).

Participants

249 NRN patients with American Spinal Injury Association Impairment Scale (AIS) level C (n = 20), D (n = 179) and (n = 50) iSCI not AIS evaluated, from February 2008 through April 2011.

Interventions

Locomotor training using body weight support and walking on a treadmill, overground and home/community practice.

Main Outcome Measure(s)

10MWT and 6MWT collected at enrollment, approximately every 20 sessions, and upon discharge.

Results

The 10MWT and 6MWT speeds were highly correlated and the 10MWT speeds were generally faster. However, the predicted 6MWT gait speed from the 10MWT, revealed increasing error with increased gait speed. Regression lines remained significantly different from lines of agreement, when the group was divided into fast (≥0.44 m/s) and slow walkers (<0.44 m/s). Significant differences between 6MWT and 10MWT gait speeds were observed across SCI-FAI walking mobility categories (Wilcoxon sign rank test p<.001), and mean speed thresholds for limited community ambulation differed for each measure. The smallest real difference for the 6MWT and 10MWT, as well as the minimally clinically important difference (MCID) values, were also distinct for the two tests.

Conclusions

While the speeds were correlated between the 6MWT and 10MWT, redundancy in the tests using predictive modeling was not observed. Different speed thresholds and separate MCIDs were defined for community ambulation for each test.     

Localisation and synthesis of α-fetoprotein in the rabbit

Summary The cellular location and sites of synthesis of -fetoprotein (AFP) in the foetal, neonatal and maternal rabbit, were studied by the fluorescent antibody technique and by culturing tissuesin vitro with labelled amino acids. AFP was found to be localised intracellularly within liver hepatocytes and yolk sac endoderm of the foetus, and within the maternal uterine epithelium. Analysis of extracts of the cultured tissues for incorporation of radioactivity into serum proteins separated by polyacrylamide gel electrophoresis or analysed by autoradiography of immuno-precipitation lines, confirmed that the foetal liver and yolk sac splanchnopleur were the principal sites of primary synthesis of AFP. Localisation of AFP in the uterine epithelium and other foetal organs was consistent with a secondary derivation from the uterine fluid or from the blood circulation. These findings are discussed in relationship to findings in man and other mammals.Supported by an award from the Medical Research Council to whom grateful acknowledgement is made.     

For the Love of Dog: The Human–Dog Bond in Rural and Remote Australian Indigenous Communities

ABSTRACT

The nature of the human–animal bond in contemporary Australian Indigenous communities is little researched, but it is essential to understand this bond in order to develop much needed appropriate animal health and management practices. A semi-structured interview format was used to elicit information on attitudes to dogs in seven Australian Indigenous communities. This explored the importance of dogs to the community and to the individual, and the balance between the positives and negatives of having dogs in the communities, with particular reference to improving dog and community health and welfare. Theme analysis of the semi-structured interview responses (n = 137) revealed a variety of attitudes to dogs within the communities. A strong theme was the importance of dogs at a community level. Many of the reasons given for the importance of dogs in the community were based on traditional cultural values or beliefs. These included dogs being necessary to guard people at night from spirits, and as part of the kin system. Further, the cultural practice of “pay-back” for wrong-doing included wrong-doing directed at dogs in all communities, even the most westernized. Occurring simultaneously with these positive attitudes, the poor health and overpopulation of dogs in the community was acknowledged and the negative effects on people's lives recognized. However, the value of the dogs to the community meant that shooting dogs without consent or poisoning them were not seen as appropriate solutions to overpopulation. Many people were prepared to euthanize some of their dogs (via an overdose of barbiturate) or have them undergo sterilization surgery. Thus, contrary to appearances from a Western perspective, the traditional Indigenous human–dog bond was found to be strong, and thus must be taken into account in developing appropriate and sustainable animal health and management practices.     

Localization and synthesis of α-fetoprotein in the chicken

Summary The localization and sites of synthesis of -fetoprotein in chick embryos throughout development have been investigated using the combined techniques of immunofluorescence microscopy and tissue culture in the presence of radiolabelled amino acids, followed by immunoautoradiographic analysis.Alpha-fetoprotein is present in a range of embryonic tissues and especially concentrated in the yolk sac, liver and connective tissue. Analysis of culture fluids revealed that the yolk sac is the major site of -fetoprotein synthesis with smaller, but significant quantities being produced by the liver.These results are discussed in relation to mammalian -fetoprotein, and the merits of the chick embryo for studies on the biological function of AFP are considered.Supported by an award from the Science Research Council, to whom grateful acknowledgement is made     

Increased Learning and Brain Long-Term Potentiation in Aged Mice Lacking DNA Polymerase μ

A definitive consequence of the aging process is the progressive deterioration of higher cognitive functions. Defects in DNA repair mechanisms mostly result in accelerated aging and reduced brain function. DNA polymerase µ is a novel accessory partner for the non-homologous end-joining DNA repair pathway for double-strand breaks, and its deficiency causes reduced DNA repair. Using associative learning and long-term potentiation experiments, we demonstrate that Polµ^−/− mice, however, maintain the ability to learn at ages when wild-type mice do not. Expression and biochemical analyses suggest that brain aging is delayed in Polµ^−/− mice, being associated with a reduced error-prone DNA oxidative repair activity and a more efficient mitochondrial function. This is the first example in which the genetic ablation of a DNA-repair function results in a substantially better maintenance of learning abilities, together with fewer signs of brain aging, in old mice.     

The Expression of Tumor Necrosis Factor-α (TNF-α) by the Intrathecal Injection of Lipopolysaccharide in the Rat Spinal Cord

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor α (TNF-α) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular site of TNF-α synthesis is still a matter of controversy. Therefore, we focused our study on TNF-α protein synthesis and expression patterns in spinal dorsal horn of naives and rats under intrathecal challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-α reached peak at 8 h. Double immunofluorescence revealed that LPS-induced expression of TNF-α exclusively located in a subpopulation of microglia, which increased at 8 h in the rat spinal dorsal horn (the injected side). Positive staining of TNF receptor 1 (TNFR1) were also found in microglia. These observations have demonstrated the production of this proinflammatory cytokine by central nerve glia especially microglia. Synthesized TNF-α might directly act on microglia via TNFR1, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation. Aiguo Shen and Dan Zhou contributed equally to this work.