Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression.

The effects of autonomic innervation of tumors on tumor growth remain unclear. Here we developed a series of genetic techniques to manipulate autonomic innervation in a tumor- and fiber-type-specific manner in mice with human breast cancer xenografts and in rats with chemically induced breast tumors. Breast cancer growth and progression were accelerated following stimulation of sympathetic nerves in tumors, but were reduced following stimulation of parasympathetic nerves. Tumor-specific sympathetic denervation suppressed tumor growth and downregulated the expression of immune checkpoint molecules (programed death-1 (PD-1), programed death ligand-1 (PD-L1), and FOXP3) to a greater extent than with pharmacological α- or ß-adrenergic receptor blockers. Genetically induced simulation of parasympathetic innervation of tumors decreased PD-1 and PD-L1 expression. In humans, a retrospective analysis of breast cancer specimens from 29 patients revealed that increased sympathetic and decreased parasympathetic nerve density in tumors were associated with poor clinical outcomes and correlated with higher expression of immune checkpoint molecules. These findings suggest that autonomic innervation of tumors regulates breast cancer progression.

Neuropharmacological lesion localization in idiopathic Horner's syndrome in Golden Retrievers and dogs of other breeds.

OBJECTIVE: To investigate whether idiopathic Horner's syndrome (HS) in Golden Retrievers is an exclusively preganglionic disorder based on denervation hypersensitivity pharmacological testing with phenylephrine. ANIMALS STUDIED: Medical records of dogs presented with HS between 2000 and 2012. Dogs presented with additional ocular or systemic signs were excluded. PROCEDURES: Clinical data examined included age, sex, duration of clinical signs, ancillary diagnostic test results, and time to mydriasis on topical ocular application of 1% phenylephrine. Lesions were diagnosed as postganglionic (mydriasis within 20 min) or preganglionic (mydriasis between 20 and 45 min). RESULTS: Medical records of 21 dogs of nine different breeds were included. An etiopathogenesis for Horner's syndrome was determined in five dogs, none of which were Golden Retrievers. All diagnoses correlated with pharmacological lesion localization. Ten Golden Retrievers were included (eight male and two female) with a mean age of 8.5 years (range: 4-13). Lesion localization was diagnosed as postganglionic in eight (mean: 10 min [range: 6-18]) and preganglionic in two Golden Retrievers (20 and 24 min). All cases were unilateral and had completely resolved within 15 weeks (range: 11-20). Recurrence was not reported in any of the patients. CONCLUSIONS: Idiopathic postganglionic HS was diagnosed in eight of 10 Golden Retrievers contradicting previous reports of a purely preganglionic localization. Etiopathogenesis of canine idiopathic HS remains to be determined; nevertheless, a vascular etiology cannot be excluded. Future studies using magnetic resonance angiography may aid in clarifying the pathogenesis.

Identification of the spinal pathways involved in the recovery of baroreflex control after spinal lesion in the rat using pseudorabies virus.

Neurons in the rostroventrolateral medulla (RVLM) mediate baroreflex regulation (BR) of spinal sympathetic preganglionic neurons. Previously, our laboratory has shown that recovery of BR occurs in the rat after spinal hemisection. (Zahner MR, Kulikowicz E, and Schramm LP. Am J Physiol Regul Integr Comp Physiol 301: R1584-R1590, 2011). The goal of these experiments was to determine whether the observed recovery of BR is mediated by the reorganization of ipsilateral pathways or by compensation by spared contralateral pathways. To determine this, we infected the left kidney in rats with the retrograde transynaptic tracer, pseudorabies virus (PRV), either 1 or 8 wk after left spinal hemisection at either T(3) or T(8), or after a sham lesion. In sham-lesioned rats, PRV infection of RVLM neurons was bilateral. In all rats with a left hemisection, regardless of the location of the lesion (T(3) or T(8)) or postlesion recovery time (1 or 8 wk), PRV infection of left RVLM neurons was significantly reduced compared with sham-lesioned rats (P < 0.05). In a separate group of rats, we performed BR tests by measuring responses of left renal sympathetic nerve activity to pharmacologically induced decreases and increases in arterial pressure. In rats with T(8) left hemisection and 8-wk recovery, BR was robust, and acute right upper thoracic hemisection abolished all BR of left renal sympathetic nerve activity. Collectively, these data suggest that the recovery of BR is not mediated by reorganization of ipsilateral bulbospinal connections, but instead by improved efficacy of existing contralateral pathways.

Sensory Guillain-Barré syndrome and related disorders: an attempt at systematization.

The possibility that some patients diagnosed with an acute sensory neuropathy could actually have Guillain-Barré syndrome (GBS) has been repeatedly advanced in the literature, but the number of cases reported is small. The reports have shown different clinical presentations and electrophysiological findings and are variously named, thus generating terminological and nosological confusion. We operatively defined sensory GBS as an acute, monophasic, widespread neuropathy characterized clinically by exclusive sensory symptoms and signs that reach their nadir in a maximum of 6 weeks without related systemic disorders and other diseases or conditions. We reviewed the literature through searches of PubMed from 1980 to March 2011 and our own files. On the basis of the size of fibers involved and the possible site of primary damage, we propose tentatively classifying sensory GBS and related disorders into three subtypes: acute sensory demyelinating polyneuropathy; acute sensory large-fiber axonopathy-ganglionopathy; and acute sensory small-fiber neuropathy-ganglionopathy.

Magnetic resonance findings in the pregeniculate visual pathways in Leber hereditary optic neuropathy.

Two relatives, a 61-year-old man and the 21-year-old grandson of his sister, suffered from bilateral visual loss and were diagnosed with Leber hereditary optic neuropathy. In both cases, the diagnosis was molecularly confirmed with the 11778 mitochondrial mutation. MRI showed increased T2 signal not only in the optic nerves and chiasm but also in the optic tracts, extending to the lateral geniculate bodies. To our knowledge, the latter finding has not been described previously.

Selected changes in spinal cord morphology after T4 transection and olfactory ensheathing cell transplantation.

Spinal cord transection at T4 results in severe damage of the nervous tissue, with impairment of motor, sensory and autonomic functions. Transplantation of olfactory ensheathing cells (OECs) has the potential to improve these functions through a number of mechanisms, which include facilitation of regeneration and neuroprotection. For cardiovascular functions, we have previously shown that OECs reduce the duration of autonomic dysreflexia, without evidence of regeneration. To further understand the mechanisms underpinning this improvement, we have studied changes in selected morphological features (cavitation, non-cavity tissue loss, morphology of sympathetic preganglionic neurons and primary afferent fibre density) in the T4-transected rat spinal cord over 9 weeks, both in control and OEC-transplanted animals. T4 transection led to a number of structural changes: gradual formation of cavities, non-cavity tissue loss, a long-term increase in soma size of sympathetic preganglionic neurons and a temporary increase in the extent of their dendritic arbours, and an increase in the density of primary afferent fibres caudal to the lesion. OECs decreased the cavitation and normalised soma size of the sympathetic preganglionic neurons below the lesion, while increasing the extent of dendritic arbours in the preganglionic neurons above the lesion. Thus the OECs may contribute to the normalisation of the dysreflexic hypertension through tissue preservation and normalisation of the morphology of the preganglionic neurons caudal to the lesion, while enhancing the input on the rostral preganglionic neurons, whose vasomotor control remains intact. We hypothesise that these changes are mediated through secretion of soluble trophic factors by the transplanted OECs.

Noxious colorectal distention in spinalized rats reduces pseudorabies virus labeling of sympathetic neurons.

The retrograde transsynaptic tracer pseudorabies virus (PRV) has been widely used as a marker for synaptic connectivity in the spinal cord. Notably, the PRV-152 construct expresses enhanced green fluorescent protein (EGFP). We recently reported a significant attenuation of PRV-152 labeling of the intermediolateral cell column (IML) and celiac ganglia after complete T4 spinal cord transection versus sham injury in rats at 96 h after PRV-152 inoculation of the left kidney. Here we found a significant increase in noxious colorectal distention (CRD)-evoked c-Fos expression in spinal cords of injured versus sham rats without PRV infection. In order to assess whether enhancing neuronal activity in spinalized rats might increase PRV-152 labeling, we subjected awake spinalized rats to 1.5 h of intermittent noxious CRD either: (1) just prior to inoculation, or (2) 96 h after inoculation (n = 3/group). Equal numbers of spinalized rats in both groups received PRV-152 inoculations without CRD (non-stimulated; n = 3/group). At 96 h post-inoculation fixed spinal cords and left celiac ganglionic tissues were assessed for the distribution and quantification of EGFP-labeled cells. The injured cohort that received CRD just prior to PRV injection showed a significant reduction in EGFP-labeled cells in both the IML and left celiac ganglion compared to non-stimulated injured rats. In contrast, the injured cohort that received CRD 96 h after PRV-152 inoculation showed no differences in EGFP-labeled cell numbers in the IML or celiac ganglia versus non-stimulated injured rats. Interestingly, microglia near c-Fos-positive cells after acute CRD appeared more reactive compared to non-stimulated spinalized rats, and activated microglial cells markedly reduce viral transduction and progression following PRV inoculation of the CNS. Hence our results imply that increased CRD-induced c-Fos expression in the injured paradigm, prior to but not after PRV injection, further attenuates PRV-152 uptake, perhaps through changes in neuronal activity and/or innate neuro-immune responses.

Olfactory ensheathing cells reduce duration of autonomic dysreflexia in rats with high spinal cord injury.

Autonomic dysreflexia is a common complication in high spinal cord injury and can result in serious consequences and death. Here we have examined the effect of acute transplantation of olfactory ensheathing cells on cardiovascular functions in rats. After T4 transection, radio-telemetric recording in conscious animals was used to study blood pressure and heart rate at rest and during autonomic dysreflexia for up to 8 weeks post-injury. Olfactory ensheathing cells from syngeneic rats were transplanted at the injury site; control animals received culture medium only. At the study end point, we examined morphometric features of sympathetic preganglionic neurons above and below the injury. T4 transection resulted in a fall in resting mean arterial pressure and an increase in resting heart rate. Colorectal distension, used to trigger autonomic dysreflexia, caused episodic hypertension and bradycardia. Although the cell transplantation had no effect on resting cardiovascular parameters, it led to a significantly faster recovery from hypertension, with the recovery time shortened by approximately 25%. The transection resulted in an increase in soma size of sympathetic preganglionic neurons above and below the injury. OEC transplantation normalised this change below the injury and increased dendritic length of preganglionic neurons above the injury, compared to controls. It has been proposed that changes in sympathetic preganglionic neurons following spinal cord transection may be related to the development of autonomic dysreflexia. Our results suggest that olfactory ensheathing cells may alter the morphology of these neurons, and hence modify their activity in the neuronal networks responsible for the dysreflexic reaction.

Periganglionic inflammation elicits a distally radiating pain hypersensitivity by promoting COX-2 induction in the dorsal root ganglion.

We have developed a model in which inflammation contiguous to and within a dorsal root ganglion (DRG) was generated by local application of complete Freund's adjuvant (CFA) to the L4 lumbar spinal nerve as it exits from the intervertebral foramen. The periganglionic inflammation (PGI) elicited a marked reduction in withdrawal threshold to mechanical stimuli and an increase in heat pain sensitivity in the ipsilateral hindpaw in the absence of any hindpaw inflammation. The pain sensitivity appeared within hours and lasted for a week. The PGI also induced a prominent increase in IL-1beta and TNF-alpha levels in the DRG and of cyclooxygenase-2 (COX-2) expression in neurons and satellite cells. A selective COX-2 inhibitor reduced the PGI-induced hyperalgesia. We also show that IL-1beta induces COX-2 expression and prostaglandin release in DRG neurons in vitro in a MAP kinase-dependent fashion. The COX-2 induction was prevented by ERK and p38 inhibitors. We conclude that periganglionic inflammation increases cytokine levels, including IL-1beta, leading to the transcription of COX-2 and prostaglandin production in the affected DRG, and thereby to the development of a dermatomally distributed pain hypersensitivity.

[Alteration of trachea and bronchi smooth muscles constriction under long inhalation of dioxide nitrogen by rats].

On insulated preparation of trachea and bronchi, change of the smooth muscles constriction was studied in rats inhaling dioxide nitrogen during 15, 30 and 60 days. The inhalation enhanced 1.5-fold and bronchi 2.5-fold responses of tracheal smooth muscles to stimulation of preganglionic nerve. The delay of the response in both events increased 1.3-fold. Long repeated stimulation caused a further increase of the constriction of tracheas and bronchi (1.32-fold). Blocking receptor with Novocain (1.0 mcg/ml) reduced the amplitude of the constriction and enhanced the response delay. After 30 days of inhalation, repeated nerve stimulations enhanced the amplitude of the constriction up to 120%, but after 60 days it reduced the amplitude of the constriction to 71 and 77% in preparations of tracheas and bronchi, respectively. In our opinion, long inhalation of dioxide nitrogen broke functioning of local lung nervous system controlling activity of the smooth muscles. The research results indicate important role of neuron functional module in pathology of the respiratory ways caused inhalation of dioxide nitrogen.

Age-related loss of cardiac vagal preganglionic neurones in spontaneously hypertensive rats.

Despite the findings that impaired vagal control of the heart rate occurs in human hypertension, leading to greater cardiovascular risk, the mechanism of this impairment is as yet unknown. Observations in humans and experiments in the spontaneously hypertensive rat (SHR) suggested that such impairment may be related to an anomaly in central vagal neurones. We therefore set out to determine whether the numbers and distribution of cardiac-projecting vagal preganglionic neurones in the medulla of adult (12 week) hypertensive SHR are different from those in young (4 week) prehypertensive SHR and in age-matched Wistar-Kyoto (WKY) rats of two age groups. The number of vagal neurones, identified by labelling with the fluorescent tracer DiI applied to the heart, was essentially similar in the three areas of the medulla analysed (dorsal vagal nucleus, nucleus ambiguus and intermediate reticular zone) in young SHR and young or adult WKY rats. In contrast, fewer vagal neurones were labelled in adult SHR compared with young SHR or WKY rats. This difference was due to highly significant reductions in vagal neurones in the dorsal vagal nucleus and nucleus ambiguus on the right side of the medulla. These observations suggest that a loss of parasympathetic preganglionic neurones supplying the heart with axons in the right vagus nerve, or a remodelling of their cardiac projections, may explain the known impairment of the baroreceptor reflex gain controlling heart rate in hypertension.

Activating transcription factor 3 immunoreactivity identifies small populations of axotomized neurons in rat cervical sympathetic ganglia after transection of the preganglionic cervical sympathetic trunk.

Activating transcription factor 3 (ATF3) has been proposed as a marker for injured neurons. Thus, while undetectable normally in sensory, motor, or sympathetic neurons, ATF3-like immunoreactivity (ATF3-IR) is readily detectable in such cells after axotomy. Here we examined ATF3-IR in the superior cervical ganglion (SCG) and the middle and inferior cervical ganglia (MICG) after transection of the predominantly preganglionic cervical sympathetic trunk (CST). The purpose of the study was to determine whether neurons in the SCG would exhibit ATF3-IR after decentralization and, if they did not, whether the induction of ATF3-IR was sensitive enough to identify the small numbers of neurons in the SCG and MICG that project their axons into the CST. Following transection of the CST, the majority of deafferented neurons in the SCG showed no ATF3-IR; however, a small group of neurons in both the SCG and MICG were labeled, and the location of the labeled neurons within these ganglia corresponded to that of neurons axotomized by this procedure. Furthermore, the ATF3-positive neurons in the MICG could be retrogradely labeled from the transected CST. In addition, a large number of smaller cells were labeled in the SCG, though not in the MICG, and some of these cells were double labeled with an antiserum to the glial protein S-100. These data indicate that, after transection of the CST, neuronal labeling in the SCG and MICG is restricted to axotomized neurons but that in addition there is extensive labeling of glial cells associated with anterograde degeneration within the SCG.

Novel repair strategies to restore bladder function following cauda equina/conus medullaris injuries.

Trauma to the thoracolumbar junction or lumbosacral spine may result in a conus medullaris or cauda equina syndrome. In both conditions, symptoms typically include paraparesis or paraplegia, sensory impairment, pain, as well as bladder, bowel, and sexual dysfunctions. We present in this review a series of neural repair strategies that have been developed to address the unique features and challenges of subjects with a conus medullaris or cauda equina syndrome. We address, in particular, neural repair strategies that may have a translational research potential to restore bladder function. Recent animal injury models have suggested that a progressive retrograde death of both autonomic and motor neurons may contribute to the neurological deficits in subjects with conus medullaris and cauda equina injuries. For subjects with acute injuries, we present novel strategies to promote neuroprotection, axonal regeneration, and functional reinnervation of the lower urinary tract. For subjects with chronic injuries, we discuss new approaches to replace lost autonomic and motor neurons. A brief discussion on a variety of outcome measures that may be suitable to evaluate the function of the lower urinary tract in rodent neural repair models is also provided.

Ectopic sympathetic preganglionic neurons maintain proper connectivity in the reeler mutant mouse.

The location of sympathetic preganglionic neurons (SPN) in the spinal cord of the reeler mouse mutant is abnormal. Instead of their normal location in the intermediolateral column, the majority of SPN in the reeler cluster around the central canal. To determine whether ectopically located SPN in the reeler form appropriate synaptic connections with their pre- and postsynaptic partners, we examined 1). whether the axons of descending neural pathways that normally terminate on SPN follow them to their ectopic location, and 2). whether the central autonomic neural circuit that controls sympathetic output to the kidney is organized normally in the reeler. Using antibodies against tyrosine hydroxylase, serotonin, neuropeptide Y, substance P and calcitonin gene-related peptide as markers for adrenergic, serotonergic and peptidergic terminals, we found that axons which normally innervate SPN follow these neurons to their ectopic spinal location in the reeler. Injection of pseudorabies virus into the kidney of wild type and reeler mutant mice revealed similar patterns of renal sympathetic and pre-sympathetic control circuits in the spinal cord, brainstem and forebrain. These results indicate that the presynaptic inputs and postsynaptic targets of SPN in the reeler are normal, despite the ectopic spinal location of their cell bodies.

Parasympathetic schwannoma of the submandibular gland.

Schwannomas of the submandibular gland have only been documented sporadically throughout the medical literature. We describe a case of schwannoma of the submandibular gland originating from the submandibular branch of the lingual nerve, which carries the preganglionic parasympathetic nerve fibers. The clinical aspect of this tumor is discussed, followed by a brief literature review.