Reg-2 expression in dorsal root ganglion neurons after adjuvant-induced monoarthritis.

Reg-2 is a secreted protein that is expressed de novo in motoneurons, sympathetic neurons, and dorsal root ganglion (DRG) neurons after nerve injury and which can act as a Schwann cell mitogen. We now show that Reg-2 is also upregulated by DRG neurons in inflammation with a very unusual expression pattern. In a rat model of monoarthritis, Reg-2 immunoreactivity was detected in DRG neurons at 1 day, peaked at 3 days (in 11.6% of DRG neurons), and was still present at 10 days (in 5%). Expression was almost exclusively in the population of DRG neurons that expresses the purinoceptor P2X(3) and binding sites for the lectin Griffonia simplicifolia IB4, and which is known to respond to glial cell line-derived neurotrophic factor (GDNF). Immunoreactivity was present in DRG cell bodies and central terminals in the dorsal horn of the spinal cord. In contrast, very little expression was seen in the nerve growth factor (NGF) responsive and substance P expressing population. However intrathecal delivery of GDNF did not induce Reg-2 expression, but leukemia inhibitory factor (LIF) had a dramatic effect, inducing Reg-2 immunoreactivity in 39% of DRG neurons and 62% of P2X(3) cells. Changes in inflammation have previously been observed predominantly in the neuropeptide expressing, NGF responsive, DRG neurons. Our results show that changes also take place in the IB4 population, possibly driven by members of the LIF family of neuropoietic cytokines. In addition, the presence of Reg-2 in central axon terminals implicates Reg-2 as a possible modulator of second order dorsal horn cells.

Axonal transport of activating transcription factor-2 is modulated by nerve growth factor in nociceptive neurons.

The aim of this study was to determine whether axonal transport of activating transcription factor-2 (ATF2) occurs in adult sensory neurons, and whether this process is under neurotrophin control. Antisera to both total ATF2 and to the activated (i.e., phosphorylated) form were used for immunocytochemistry and Western blotting. ATF2 was localized to predominantly nociceptive dorsal root ganglion cells in adult rats and shown to accumulate proximal and distal to a sciatic nerve ligature as a result of axonal transport. Subcutaneous injection of nerve growth factor (NGF) decreased the levels of fast retrograde axonal transport of activated ATF2 by 97% (p < 0.05) and elevated levels of retrograde axonal transport of total ATF2 by twofold (p < 0.02). In contrast, blocking endogenous NGF using an anti-NGF antibody induced an elevation in retrograde axonal transport of activated ATF2 of 4. 5-fold (p < 0.05) and decreased retrograde axonal transport of total ATF2 by 72% (p < 0.05). NGF or anti-NGF treatment had no effect on the anterograde transport levels of total or activated ATF2. This study shows that signaling by target-derived NGF to the cell bodies of sensory neurons consists, in part, of the modulation of levels and activation status of a retrogradely transported transcription factor, ATF2.

Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy.

Aberrant neurofilament phosphorylation occurs in many neurodegenerative diseases, and in this study, two animal models of type 1 diabetes--the spontaneously diabetic BB rat and the streptozocin-induced diabetic rat--have been used to determine whether such a phenomenon is involved in the etiology of the symmetrical sensory polyneuropathy commonly associated with diabetes. There was a two- to threefold (P < 0.05) elevation of neurofilament phosphorylation in lumbar dorsal root ganglia (DRG) of diabetic rats that was localized to perikarya of medium to large neurons using immunocytochemistry. Additionally, diabetes enhanced neurofilament M phosphorylation by 2.5-fold (P < 0.001) in sural nerve of BB rats. Neurofilaments are substrates of the mitogen-activated protein kinase (MAPK) family, which includes c-jun NH2-terminal kinase (JNK) or stress-activated protein kinase (SAPK1) and extracellular signal-regulated kinases (ERKs) 1 and 2. Diabetes induced a significant three- to fourfold (P < 0.05) increase in phosphorylation of a 54-kDa isoform of JNK in DRG and sural nerve, and this correlated with elevated c-Jun and neurofilament phosphorylation. In diabetes, ERK phosphorylation was also increased in the DRG, but not in sural nerve. Immunocytochemistry showed that JNK was present in sensory neuron perikarya and axons. Motoneuron perikarya and peroneal nerve of diabetic rats showed no evidence of increased neurofilament phosphorylation and failed to exhibit phosphorylation of JNK. It is hypothesized that in sensory neurons of diabetic rats, aberrant phosphorylation of neurofilament may contribute to the distal sensory axonopathy observed in diabetes.

TrkA immunoreactive neurones in the rat spinal cord.

We report the presence in rat spinal cord of a novel neuronal system expressing tyrosine kinase receptor (trkA), the high affinity receptor for nerve growth factor (NGF). TrkA immunoreactive cell bodies were observed in the intermediate grey matter of the spinal cord and were classified into three main groups: central canal cells located dorsolateral to the aqueduct, partition cells located between lamina X, and the lateral border of the intermediate grey, and a morphologically heterogeneous group which included large cells located near the lateral border. In situ hybridization confirmed that cells in all these areas express trkA mRNA. Combined immunofluorescence and retrograde Fluoro-Gold labelling was used to further characterise the projections and neurotransmitter profile of the trkA cells. Although often located in the vicinity of preganglionic cell groups, trkA immunoreactive cells are not themselves preganglionic. Rather, the central canal and partition cells belong to a neurochemically complex cholinergic propriospinal system. Many partition cells coexpress trkA, choline acetyltransferase (ChAT), the low affinity neurotrophin receptor, p75, and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). In contrast, trkA immunoreactive central canal cells express ChAT, but do not express p75 and only a subpopulation express NADPH-d. The large trkA immunoreactive cells located on the lateral border do not express ChAT. TrkA immunoreactive fibres were also present and were located in the dorsal horn, in the dorsal columns, and in a bundle ventral to the aqueduct. However, double labelling revealed that the trkA immunoreactive fibres are not intrinsic but are primary afferent in origin and coexpress p75. The location of this novel trkA neuronal system is consistent with it having a role in the segmental integration of autonomic outflow. NGF could affect this system by modulating neuronal phenotype and/or synaptic efficacy.

Distribution of messenger RNAs encoding enkephalin, substance P, somatostatin, galanin, vasoactive intestinal polypeptide, neuropeptide Y, and calcitonin gene-related peptide in the midbrain periaqueductal grey in the rat.

The midbrain periaqueductal grey matter (PAG) has numerous functional roles that include mediating nociceptive inhibition and integrating behavioural and physiological responses to potentially threatening or stressful stimuli. Underlying these behaviours is the diverse interconnectivity of this region, and it is possible that neurochemical subdivisions within the PAG reflect the functional properties of the different PAG regions. In this study, using in situ hybridization, we have investigated the distribution in the rat PAG of the messenger ribonucleic acids (mRNAs) encoding seven neuropeptides: enkephalin (ENK), substance P (SP), somatostatin (SST), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP). Each peptide mRNA had a distinct topographical distribution in the PAG. Preproenkephalin A (ENK) mRNA-expressing cells were found at all levels of the PAG in three distinct longitudinal columns. Preprotachykinin A (SP)-expressing cells were found at all levels of the PAG, principally in the Edinger-Westphal nucleus and the lateral and dorsal PAG. There was a column of neurons producing mRNA-encoding somatostatin that extended along the rostrocaudal extent of the ventrolateral PAG; there were also labelled cells in the dorsal and dorsolateral subdivisions at some levels of the PAG. Galanin mRNA-producing neurones were limited to the dorsal raphe nucleus and to a second population in the ventral border of the aqueduct. VIP mRNA-producing neurones were found in very localized regions of the PAG, including the cell-sparse region immediately ventral to the aqueduct and the ventral part of the dorsal raphe nucleus. NPY mRNA-producing neurones were localized mainly in some cells of the Edinger-Westphal nucleus and dorsal raphe nucleus. CGRP mRNA-expressing neurons were limited to the oculomotor and trochlear nucleus. The results showed a topographical distribution of neuropeptides over the rostrocaudal extent of the PAG that is compatible with the emerging theory that the anatomical and functional specificity of the PAG is expressed in the form of longitudinally arranged neuronal columns that extend for varying distances along the rostrocaudal axis of the midbrain PAG.

Distribution of the voltage-dependent calcium channel alpha(1A) subunit throughout the mature rat brain and its relationship to neurotransmitter pathways.

The alpha(1) subunit provides both the voltage-sensing mechanism and the ion pore of voltage-dependent calcium channels. Of the six classes of alpha(1) subunit cloned to date, alpha)1A) is the subject of debate in terms of its functional correlate, although it is generally thought to encode voltage-dependent calcium channels of the omega-agatoxin IVA-sensitive, P/Q type. In the present study, an alpha(1A)-specific riboprobe and antibody were used with in situ hybridisation and immunohistochemical techniques to localise alpha(1A) messenger ribonucleic acid transcripts and subunit protein throughout the mature rat brain. Dual localisation of alpha(1A) protein and markers for acetylcholine, catecholamines, and 5-hydroxytryptamine have also been performed in a number of discrete areas. Abundant and widespread distribution of alpha(1A) protein was found, with immunoreactivity occurring both in cell bodies and as punctate staining in areas of neuronal processes. Several associations were noted across alpha(1A) localisation, defined neuroanatomical regions, and neurotransmitter systems. However, alpha(1A) expression was not confined to loci corresponding to any one neurotransmitter type, although a high level of expression was observed in cholinergic neurones. The distribution of the alpha(1A) subunit in the rat corresponded well with the limited human mapping data that are available.

Neuronal calcium sensor-1 is expressed by dorsal root ganglion cells, is axonally transported to central and peripheral terminals, and is concentrated at nodes.

Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily which has been implicated in the modulation of a number of neuronal functions. In this study we have examined the expression of NCS-1 in adult rat dorsal root ganglion (DRG) neurons. NCS-1 immunoreactivity was present in most DRG neurons, including many calcitonin gene-related peptide (CGRP) expressing ones. NCS-1 showed some colocalization with the synaptic vesicle protein synaptophysin and underwent both anterograde and retrograde axonal transport. NCS-1 immunoreactivity was also present in the dorsal horn of the spinal cord, and in peripheral cutaneous terminals innervating blood vessels, where it was coexpressed with CGRP. In addition, NCS-1 in peripheral nerves was concentrated at nodes and adjoining paranodes. These results suggest novel roles for NCS-1, particularly in relation to channel function at nodes and to the peripheral release of vasoactive peptides.

Effects of glial cell line-derived neurotrophic factor on axonal growth and apoptosis in adult mammalian sensory neurons in vitro.

The effects of glial cell line-derived neurotrophic factor on axonal outgrowth and apoptosis were studied in vitro using explanted dorsal root ganglia-peripheral nerve preparations of adult mice. In gels of matrigel or collagen type 1, glial cell line-derived neurotrophic factor increased both the numbers and lengths of axons growing out of explanted preparations, although less effectively than nerve growth factor. Stimulation of axonal outgrowth by glial cell line-derived neurotrophic factor was unaffected by K252a, a protein kinase inhibitor which blocks the effects of nerve growth factor and other neurotrophins acting through trk receptors. To determine the phenotype of the axons responding to glial cell line-derived neurotrophic factor, preparations were stained using antibodies to trkA, calcitonin gene-related peptide, 200,000 mol. wt phosphorylated neurofilaments (monoclonal antibody RT97) and the lectin Bandeiraea simplicifolia 1B4. RT97 recognizes large diameter neurons whilst 1B4 labels small diameter neurons which broadly do not express neurotrophin receptors. In preparations cultured with glial cell line-derived neurotrophic factor, significant increases in the numbers of outgrowing axons labelled with RT97 and 1B4 were observed but the numbers of calcitonin gene-related peptide-positive axons were not significantly increased and their staining intensity was generally faint. In separate preparations it was found that in the presence of glial cell line-derived neurotrophic factor, the majority of the 1B4 labelled axons were trkA negative, indicating that this factor can stimulate axonal growth in this population of neurons which do not respond to the neurotrophins. Spontaneous apoptosis in neurons and satellite cells occurs in explanted preparations of the type used in the present investigations, but in cryostat sections of preparations cultured in the presence of glial cell line-derived neurotrophic factor, the incidence of apoptosis was lower than in control preparations which had been cultured in the absence of this factor. This suggests that glial cell line-derived neurotrophic factor may promote survival of some adult sensory neurons in vitro.

The effects of ethane dimethane sulphonate (EDS) on bilaterally cryptorchid rat testes.

A single dose of ethane dimethane sulphonate (EDS) given to adult male rats has a specific destructive effects on Leydig cells, which are removed from the intertubular area by macrophages. This is associated with a decrease in testosterone (T) and a rise in serum FSH and LH for 21-28 days after EDS. Recovery of Leydig cells occurs from connective tissue precursors. This study uses the bilaterally cryptorchid rat to investigate the influence of local factors from the seminiferous tubules on the restoration of Leydig cell morphology and function. Morphometric data of intertubular tissue, hCG binding, serum T, FSH and LH levels all indicate that the Leydig cell population in cryptorchid testes is more rapidly restored from connective tissue cells when compared to the normal situation. The precise mechanism for this faster recovery will require further study.

A combined in situ hybridization and immunofluorescence procedure allowing visualisation of peptide mRNA and serotonin in single sections.

We describe a novel procedure for combining immunocytochemistry with in situ hybridisation. In contrast to previously published procedures, the technique involves immunofluorescence followed by in situ hybridization and is particularly suitable for antigens which are labile or sensitive to in situ hybridization processing. We have evaluated the technique using 5-hydroxytryptamine (5-HT, serotonin) immunofluorescence and neuropeptide in situ hybridization employing 35S-labelled oligonucleotide probes. Successful double labelling was obtained and showed that galanin messenger RNA (mRNA) is expressed by 5-HT immunoreactive cells in the dorsal raphe nucleus of the rat. In contrast, somatostatin mRNA in the same region is expressed by a separate non-serotonergic cell population. Double-labelled preparations produced using this technique can be conveniently viewed using epipolarised combined with epifluorescent illumination. Careful analysis of procedural variables revealed that it is not possible to carry out high-sensitivity 5-HT immunocytochemistry following in situ hybridization. The immunostaining is much poorer on slide-mounted sections than on free-floating sections, and 5-HT appears to be lost during the in situ hybridization steps of dehydration/delipidation and incubation in hybridization buffer. The procedure we describe avoids these problems but with a slight loss of in situ hybridization sensitivity.

Serotonin innervation of enkephalin containing neurones in the rat spinal trigeminal nucleus.

Light and electron microscopic immunocytochemistry was used to examine the serotonin (5-HT) innervation of the rat spinal trigeminal subnucleus caudalis. 5-HT-immunoreactive fibres form a dense plexus in lamina I and outer lamina II and synapse with the cell bodies and proximal dendrites of local neurones. Light microscopic double labelling revealed that the 5-HT axons contact enkephalin immunoreactive neurones in both laminae. The 5-fHT electron microscopic results indicate that at least some of these contacts are likely to be synapses. 5-HT axons are therefore in a position to exert a direct action on enkephalinergic interneurones and this may contribute to the analgesic actions of the 5-HT system.

Serotonergic terminals express a growth associated protein (GAP-43) in the adult rat spinal cord.

Dual colour immunofluorescence has been used to compare the distribution of serotonin (5-hydroxytryptamine, 5-HT) and GAP-43 in the adult rat. GAP-43 immunostaining was observed in all spinal cord regions containing 5-HT immunoreactivity. 5-HT and GAP-43 double labelled fibres and varicosities were present and were most evident around motoneurones, in lamina X, and in the intermediolateral cell column. Single labelled GAP-43 fibres and varicosities were also observed and were the dominant population in the dorsal horn and in certain fibre tracts. We conclude that the 5-HT system is one of a small number of spinal cord systems that express high levels of GAP-43 in the adult.

Responses of different types of ox muscle to electrical stimulation.

The effects of electrical stimulation on fall in pH upon stimulation (ΔpH), rate of pH fall (dpH/dt at 35°C, cold shortening and muscle ultrastructure were investigated for the Cutaneus trunci (predominantly fast-twitch glycolytic fibres), the Masseter and Diaphragm (predominantly slow-twitch oxidative fibres) and the Sternomandibularis and Longissimus dorsi (bot fast- and slow-twitch fibres) of the ox. The Masseter and Diaphragm showed a small ΔpH and no increase in dpH/dt upon stimulation. Stimulation produced supercontracture but no tearing of the fibres throughout all of the Masseter. Stimulation of the Cutaneus trunci resulted in a significantly increased ΔpH and dpH/dt, loss of glycogen, mitochondrial swelling but no gross sarcomere changes. The Longissimus dorsi and Sternomandibularis had a moderate ΔpH and an increase in dpH/dt intermediate between that of the Masseter and the Cutaneus trunci. The Longissimus dorsi showed supercontracture, but the Sternomandibularis did not. Cold shortening responses at 2°C and 0°C were virtually unaffected by stimulation, being greatest in the Masseter and Diaphragm and least in the Cutaneus trunci. All muscles showed significantly greater shortenings at 0°C than 2°C. Stimulation of the Cutaneus trunci did not affect the tenderness of the cooked meat, but the toughness increased dramatically in cold-shortened Cutaneus muscle, regardless of stimulation. The Cutaneus trunci least requires stimulation as it does not cold shorten appreciably and therefore early rigor would confer no advantage. The Masseter and Diaphragm have a fast dpH/dt and therefore would enter rigor early. Mixed muscles apparently have the combined, least desirable characteristics of the muscle fibre types-i.e. their rate of rigor development is slow and they cold shorten. Electrical stimulation confers a significant advantage by mitigating these mixed-muscle characteristics in carcass muscles.

Risk factors associated with development of pelvic canal stenosis secondary to sacroiliac separation: 84 cases (1985-1995).

OBJECTIVE: To measure pelvic canal diameter in dogs from a ventrodorsal radiographic view of the pelvic region, to define a normal pelvic canal diameter, to evaluate risk factors associated with stenosis of the pelvic canal secondary to sacroiliac separation, and to determine clinical signs associated with pelvic canal stenosis. DESIGN: Retrospective case series. ANIMALS: 84 case-group and 46 control-group dogs. PROCEDURE: Medical records and radiographs of dogs with conditions unrelated to pelvic fracture (control group) and dogs with sacroiliac separation (case group) in which radiographs were obtained before surgery, after surgery, or after fracture healing were reviewed. Discriminant analysis was used to determine a normal pelvic canal diameter. An ANOVA and Dunnett's two-sided test were used to determine factors associated with pelvic canal stenosis. RESULTS: Pelvic canal diameter ratio determined from control-group dogs was > or = 1.1. Pelvic canal diameter ratios were significantly less for case-group dogs on radiographs obtained before surgery and after fracture healing than for control-group dogs, regardless of fracture type or treatment, except for dogs with ilial fractures treated conservatively. Pelvic canal diameter ratios did not differ for case-group dogs on radiographs obtained after surgery from those for control-group dogs, except when ilial fractures were surgically reduced. None of the dogs had clinical signs associated with pelvic canal stenosis. CLINICAL IMPLICATIONS: Pelvic canal diameter in dogs can be determined from a ventrodorsal radiographic view of the pelvic region. Dogs with pelvic fractures that have a normal pelvic canal diameter before surgery tend to have a normal pelvic canal diameter after fracture healing.

Effect of perioperative prophylactic antimicrobial treatment in dogs undergoing elective orthopedic surgery.

OBJECTIVE: To determine whether perioperative antimicrobial prophylaxis would reduce incidence of postoperative infection among dogs undergoing elective orthopedic procedures. DESIGN: Randomized, controlled, blinded, intention clinical trial. ANIMALS: Dogs of any breed, sex, or age undergoing elective orthopedic surgery at a veterinary teaching hospital. PROCEDURES: Dogs were randomly assigned to 1 of 3 groups: treatment with saline solution, treatment with potassium penicillin G, and treatment with cefazolin. Treatments were intended to be administered within 30 minutes prior to surgery; a second dose was administered if surgery lasted > 90 minutes. Dogs were monitored for 10 to 14 days after surgery for evidence of infection. RESULTS: After the first 112 dogs were enrolled in the study, it was found that infection rate for control dogs (5/32 dogs) was significantly higher than the rate for dogs treated with antimicrobials (3/80 dogs). Therefore, no more dogs were enrolled in the study. A total of 126 dogs completed the study. Monte Carlo simulations indicated that compared with dogs that received antimicrobials prophylactically, dogs that received saline solution developed infections significantly more frequently. Difference in efficacy, however, was not observed between the 2 antimicrobial drugs used. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that perioperative antimicrobial prophylaxis decreased postoperative infection rate in dogs undergoing elective orthopedic surgery, compared with infection rate in control dogs. Cefazolin was not more efficacious than potassium penicillin G in these dogs.

Recovery of the lost good object.

A significant occurrence in the treatment of some patients involves the recovery of the lost good object, that is, recovering a preverbal experience of the "good mother." Such an experience can be a turning point in treatment, because it often solidifies the therapeutic alliance and thus facilitates a positive outcome. Although psychotherapy or psychoanalysis cannot make up for early trauma or parental failure, it facilitates the acquisition of positive memories that enable the patient to reinvest in the world and in new objects in the environment. This thesis is illustrated by material from the author's own analysis, as well as from three case examples.

Preventing staff-patient sexual relationships.

Just as the awareness of incest and childhood sexual abuse has grown in recent years, so too has knowledge of sexual exploitation of patients by psychotherapists and other mental health professionals. Almost no one, however, has discussed the prevalence of this problem in the hospital setting. The authors were members of a study group that thoroughly reviewed reported incidents in their own institution, leading to the recommendation that education is the best preventive measure currently available. The authors discuss fundamental issues involved in such a study, summarize profiles of patients and staff members who may be vulnerable to sexual involvement, and describe the main components of a preventive educational approach.

Activation transcription factor-3 activation and the development of spinal cord degeneration in a rat model of amyotrophic lateral sclerosis.

It has been reported that an early activation of glial fibrillary acid protein (GFAP) in astroglial cells occurs simultaneously in peripheral nerves and spinal cord from the G93A SOD1 mouse model of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder. In ALS, the contribute to the pathological process of different cell types varies according to the disease stage, with a florid immune response in spinal cord at end stage disease. In this study, we have mapped in different anatomical sites the process of disease-induced functional perturbation from a pre-symptomatic stage using a marker of cellular distress expressed in neurons and glial cells, the activating transcription factor 3 (ATF-3), and applied large-scale gene expression analysis to define the pattern or transcriptional changes occurring in spinal cord from the G93A SOD1 rat model of ALS in parallel with ATF-3 neuronal activation. From the disease onset onward, transgenic lumbar spinal cord displayed ATF-3 transcriptional regulation and motor cells immunostaining in association with the over-expression of genes promoting cell growth, the functional integrity of cell organelles and involved in the modulation of immune responses. While spinal cord from the pre-symptomatic rat showed no detectable ATF-3 transcriptional regulation, ATF-3 activation was appreciated in large size neurofilament-rich, small size non-peptidergic and parvalbumin-positive neurons within the dorsal root ganglia (DRG), and in ventral roots Schwann cells alongside macrophages infiltration. This pattern of peripheral ATF-3 activation remained detectable throughout the disease process. In the G93A SOD1 rat model of ALS, signs of roots and nerves subtle distress preceded overt clinical-pathological changes, involving both glial cells and neurons that function as receptors of peripheral sensory stimuli from the muscle. In addition, factors previously described to be linked to ATF-3 activation under various experimental conditions of stress, become switched on in spinal cord from the end-stage transgenic rat model of ALS.