Frequency dependent, reversible focused ultrasound suppression of evoked potentials in the reflex arc in an anesthetized animal.

OBJECTIVE: To investigate the potential for noninvasive low frequency, low intensity ultrasound to suppress evoked potentials in the reflex arc neural pathway in anesthetized animal. MATERIAL AND METHODS: Single unit Electromyographic recordings of gastrocnemius muscle activity were obtained in response to electrical activation of the sciatic nerve, in anesthetized animal, with and without US stimulation. Reflex related potentials were evoked via electrical stimulation and low-intensity, low-frequency ultrasound was applied to the sciatic nerve. Electromyogram (EMG) was recorded from the gastrocnemius muscle and amplitude and area under the curve were analyzed to determine the effect of ultrasound stimulation on potentials in the reflex arc. Thermal imaging was used to assess thermal effects of US stimulation and immunohistochemical was performed post-US stimulation assess potential damage to the nerve. RESULTS: Our results show a drop in electromyogram amplitudes as high as 20%, and a drop in areas under the curve as high as 23%, with greater effects at lower frequencies (200 kHz) and lower acoustic intensities. The suppression of EMG scales with the magnitude of the electrical stimuli. Also, our results demonstrated transient reversibility of US suppression and our experiments eliminated thermal effects and mechanical and thermal damage. CONCLUSION: The non-invasiveness of US stimulation and its inhibitory and reversible effects emphasize the potential of US as a therapeutic modality and clinical tool for suppression of neural potentials in the reflex arc such as the case for pain treatments. The study lays the ground for potential applications of US stimulation in pain treatment.

Role of sympathetic nervous system in rat model of chronic visceral pain.

BACKGROUND: Changes in central pain modulation have been implicated in generalized pain syndromes such as irritable bowel syndrome (IBS). We have previously demonstrated that reduced descending inhibition unveils a role of sympathoneuronal outflow in decreasing peripheral sensory thresholds, resulting in stress-induced hyperalgesia. We investigated whether sympathetic nervous system (SNS) exacerbation of pain sensation when central pain inhibition is reduced is relevant to chronic pain disorders using a rat colon irritation (CI) model of chronic visceral hypersensitivity with hallmarks of IBS. METHODS: Rats were treated to a series of colorectal balloon distensions (CRD) as neonates resulting in visceral and somatic hypersensitivity and altered stool function that persists into adulthood. The visceral sensitivity was assessed by recording electromyographic (EMG) responses to CRD. Somatic sensitivity was assessed by paw withdrawal thresholds to radiant heat. The effects on the hypersensitivity of (i) inhibiting sympathoneuronal outflow with pharmacological and surgical interventions and (ii) enhancing the outflow with water avoidance stress (WAS) were tested. KEY RESULTS: The alpha2-adrenergic agonist, clonidine, and the alpha1-adrenergic antagonist, prazosin, reduced the visceral hypersensitivity and WAS enhanced the pain. Chemical sympathectomy with guanethidine and surgical sympathectomy resulted in a loss of the chronic visceral hypersensitivity. CONCLUSIONS & INFERENCES: The results support a role of the SNS in driving the chronic visceral and somatic hypersensitivity seen in CI rats. The findings further suggest that treatments that decrease sympathetic outflow or block activation of adrenergic receptors on sensory nerves could be beneficial in the treatment of generalized pain syndromes.

Comparison of metabolic and neuropathy profiles of rats with streptozotocin-induced overt and moderate insulinopenia.

To assess the relative roles of insulinopenia, hyperglycemia and dyslipidemia in pathogenesis of diabetic neuropathy, we compared plasma insulin, glucose and lipid metabolism and peripheral nerve function in rats with streptozotocin (STZ)-induced overt and moderate insulinopenia (hyperglycemic, STZ-HG; random glucose>11 mM and normoglycemic, STZ-NG rats). While being slightly insulinopenic, STZ-NG rats are metabolically not different from control, naive animals, by having normal glucose tolerance and normal levels of plasma glucose, glycated HbA1c, cholesterol and triglycerides. Two weeks following injection of STZ, STZ-HG but not STZ-NG rats had suppressed motor nerve conduction velocity, F-wave prevalence, withdrawal responses to heat and von Frey filament stimuli. In apparent correlation with plasma insulin level, both STZ-HG and -NG rats manifested exaggerated responses in paw pressure and colorectal distension tests. These data suggest that insulinopenia may play a leading role in the diabetic impairment of deep muscle and visceral afferent pathways while hyperglycemia/dyslipidemia may represent a key requirement for the onset and progression of electrophysiological nerve impairment and loss of superficial heat and tactile perception. STZ-NG rats offer a convenient model for the investigation of the short-term effects of insulinopenia on peripheral nerve function.

Importance of neural mechanisms in colonic mucosal and muscular dysfunction in adult rats following neonatal colonic irritation.

Previous studies have shown that early life trauma induced by maternal separation or colonic irritation leads to hypersensitivity to colorectal distension in adulthood. We tested the hypothesis that repetitive colorectal distension in neonates leads to abnormalities in colonic permeability and smooth muscle function in the adult rat. In neonatal rats, repetitive colorectal distension was performed on days 8, 10, and 12. As adults, stool consistency was graded from 0 (formed stool) to 3 (liquid stool). Colonic tissue was isolated for histology and myeloperoxidase levels. The colonic mucosa was placed in modified Ussing chambers for measurements of permeability and short-circuit current responses to forskolin, electrical field stimulation, and carbachol. Segments of colonic musculature were placed in organ baths and contractile response to potassium chloride, electrical field stimulation, and carbachol were determined. In adult rats that experienced neonatal colonic irritation, no significant changes in colonic histology or myeloperoxidase activity were observed; however, stool consistency scores were increased. Mucosal permeability, measured as an increase in basal conductance, was significantly increased but no changes in short-circuit current responses were observed. In adulthood, rats that underwent colorectal distension as neonates exhibited an elevated smooth muscle contractile response to potassium chloride, but no changes in response to electrical field stimulation or carbachol. In summary, neonatal colonic irritation, shown previously to produce colonic hypersensitivity, leads to significant alterations in colonic mucosal and smooth muscle function characterized by loose stools, increased mucosal permeability, and increased smooth muscle contractility in the absence of colon inflammation in adulthood.

Pre-anesthetic presence of an injured dam influences pups' locomotor behavior during emergence from anesthesia in rats.

BACKGROUND: Pre-anesthetic mother-infant interaction is an important factor for smooth emergence of pediatric anesthesia. In many mammalian species, disruptions of the mother-infant relationship cause psychological and behavioral changes. This study was to investigate whether or not pre-anesthetic presence of an injured dam has an impact on locomotor behavior of rat pups. METHODS: We used a video-tracking system to test the effects of pre-anesthetic relations between pups and their dams on pups' locomotor behavior during emergence from general anesthesia, in 40 3-week-old Sprague-Dawley male rats. Pups were divided into two groups: pups housed with a dam (n = 20) and those housed with an injured dam (n = 20). Pups were anesthetized with 1.2% halothane for 30 min. At emergence, we recorded their locomotor behavior for 15 min. RESULTS: Pre-anesthetic manipulation to dams significantly increased the distance traveled by pups. However, the manipulation did not cause any difference in the maximum velocity. CONCLUSION: Pre-anesthetic presence of an injured dam influenced pups' locomotor behavior at emergence from anesthesia.

Pre-anesthetic maternal separation increases pups' locomotor behavior during emergence from anesthesia in rats.

BACKGROUND: Problematic behavior at emergence from anesthesia in children, partly linked with maternal separation, is a major problem in pediatric anesthesia. In humans, as well as in many other mammalian species, such separation causes psychological and behavioral changes. This study was to investigate whether or not pre-anesthetic maternal separation has a similar effect on rat pups. METHODS: This study was conducted on 66 3-week-old Sprague-Dawley male rats. The rats were divided into two groups; pups housed with a dam (n = 33) and those housed without (n = 33). Pups were anesthetized with 1.2% halothane for 30 min. Afterwards we recorded their locomotor behavior at emergence from general anesthesia using a video tracking system. RESULTS: Pre-anesthetic maternal separation significantly increased the maximum velocity and the distance traveled by pups at the emergence. CONCLUSION: Pre-anesthetic maternal separation influenced pups' locomotive behavior at emergence.

Cerebellar cortical stimulation increases spinal visceral nociceptive responses.

The role of the cerebellum in modulating nociceptive phenomena is unclear. In this study, we focus on the effects of cerebellar cortical stimulation on the responses of midline neurons of the lumbosacral spinal cord to graded nonnoxious and noxious visceral (colorectal distension) as well as somatic (brush, pressure, pinch) stimuli. Extracellular recording was used for the isolation and recording of spinal nociceptive neurons, while electrical current pulses and chemical injection of D, L-homocysteic acid were used to stimulate the cortex of the posterior cerebellar vermis. Cerebellar cortical stimulation increased the responses of all isolated cells to colorectal distension, whereas the effect on the responses to somatic stimuli was variable. These findings indicate that the posterior cerebellar vermis may exert a pro-nociceptive effect on spinal visceroceptive neurons.

A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development.

BACKGROUND & AIMS: The irritable bowel syndrome (IBS) is a common disorder characterized by abdominal pain in the setting of altered perception of viscerosensory stimuli. This so-called visceral hyperalgesia occurs in the absence of detectable organic disease in the peripheral organs and may cause normal or physiologic contractions to be perceived as painful. Although the pathogenesis of IBS remains speculative and is probably multifactorial, a prevailing paradigm is that transient noxious events lead to long-lasting sensitization of the neural pain circuit, despite complete resolution of the initiating event. METHODS: Neonatal male Sprague-Dawley rats received either mechanical or chemical colonic irritation between postnatal days 8 and 21 and were tested when they became adults. The abdominal withdrawal reflex and the responses of viscerosensitive neurons were recorded during colon distention. RESULTS: Colon irritation in neonates, but not in adults, results in chronic visceral hypersensitivity, with characteristics of allodynia and hyperalgesia, associated with central neuronal sensitization in the absence of identifiable peripheral pathology. CONCLUSIONS: These results concur largely with observations in patients with IBS, providing a new animal model to study IBS and validating a neurogenic component of functional abdominal pain that encourages novel approaches to health care and research.

Comparative study of viscerosomatic input onto postsynaptic dorsal column and spinothalamic tract neurons in the primate.

The purpose of the present investigation was to examine, in the primate, the role of the postsynaptic dorsal column (PSDC) system and that of the spinothalamic tract (STT) in viscerosensory processing by comparing the responses of neurons in these pathways to colorectal distension (CRD). Experiments were done on four anesthetized male monkeys (Macaca fascicularis). Extracellular recordings were made from a total of 100 neurons randomly located in the L(6)-S(1) segments of the spinal cord. Most of these neurons had cutaneous receptive fields in the perineal area, on the hind limbs or on the rump. Forty-eight percent were PSDC neurons activated antidromically from the upper cervical dorsal column or the nucleus gracilis, 17% were STT neurons activated antidromically from the thalamus, and 35% were unidentified. Twenty-one PSDC neurons, located mostly near the central canal, were excited by CRD and three were inhibited. Twenty-four PSDC neurons, mostly located in the nucleus proprius, did not respond to CRD. Of the 17 STT neurons, 7 neurons were excited by CRD, 4 neurons were inhibited, and 6 neurons did not respond to CRD. Of the unidentified neurons, 23 were excited by CRD, 7 were inhibited, and 5 did not respond. The average responses of STT and PSDC neurons excited by CRD were comparable in magnitude and duration. These results suggest that the major role of the PSDC pathway in viscerosensory processing may be due to a quantitative rather than a qualitative neuronal dominance over the STT.

A visceral pain pathway in the dorsal column of the spinal cord.

A limited midline myelotomy at T10 can relieve pelvic cancer pain in patients. This observation is explainable in light of strong evidence in support of the existence of a visceral pain pathway that ascends in the dorsal column (DC) of the spinal cord. In rats and monkeys, responses of neurons in the ventral posterolateral thalamic nucleus to noxious colorectal distention are dramatically reduced after a lesion of the DC at T10, but not by interruption of the spinothalamic tract. Blockade of transmission of visceral nociceptive signals through the rat sacral cord by microdialysis administration of morphine or 6-cyano-7-nitroquinoxaline-2,3-dione shows that postsynaptic DC neurons in the sacral cord transmit visceral nociceptive signals to the gracile nucleus. Retrograde tracing studies in rats demonstrate a concentration of postsynaptic DC neurons in the central gray matter of the L6-S1 spinal segments, and anterograde tracing studies show that labeled axons ascend from this region to the gracile nucleus. A similar projection from the midthoracic spinal cord ends in the gracile and cuneate nuclei. Behavioral experiments demonstrate that DC lesions reduce the nocifensive responses produced by noxious stimulation of the pancreas and duodenum, as well as the electrophysiological responses of ventral posterolateral neurons to these stimuli. Repeated regional blood volume measurements were made in the thalamus and other brain structures in anesthetized monkeys in response to colorectal distention by functional MRI. Sham surgery did not reduce the regional blood volume changes, whereas the changes were eliminated by a DC lesion at T10.

Epigastric antinociception by cervical dorsal column lesions in rats.

BACKGROUND: Previous clinical evidence and electrophysiologic studies in the authors' laboratory have implicated the dorsal column (DC) as an important pathway for the transmission of visceral colorectal pain. This study examined, behaviorally and electrophysiologically, the role of the DC in mediating epigastric nociception using a visceral pain model involving duodenal distension in rats. METHODS: For behavioral testing, the writhing-like responses produced in awake rats by graded intraduodenal balloon distension (0.1 to 0.7 ml) were tested. A DC mechanical lesion at the C2 level or a sham operation (SH, same spinal cord segment exposed but no DC lesion) was performed. The writhing-like responses to duodenal distension were tested again and the rats were compared with other rats with no lesions and with SH rats. For electrophysiologic testing, the extracellular activity of single neurons was recorded in the ventrobasal nucleus of the thalamus in anesthetized rats. The ventrobasal cells that responded to duodenal distension were tested further with this visceral stimulus before and after a lesion of the DC. RESULTS: The mechanical DC lesion significantly reduced the intensity of the writhing-like responses and increased the threshold volume that would elicit writhing-like responses compared with rats with no lesions and SH rats without any observable neurologic deficit. A lesion of the DC also significantly reduced the responses of ventrobasal cells to duodenal distension. CONCLUSIONS: The DC plays an important role in signaling epigastric nociception in this experimental model. A mechanical DC lesion can produce significant visceral antinociception in rats.

A role for the dorsal column in nociceptive visceral input into the thalamus of primates.

A possible role of the dorsal column (DC) in the processing of visceral pain has gained attention after studies in the rat have revealed that the DC transmits a major part of the pelvic visceral nociceptive input from the colon into the thalamus. Furthermore, clinical interventions aimed at interrupting ascending DC axons near the midline were successful in relieving the pain suffered by patients with cancer of the pelvic organs. The purpose of this study was to check whether a DC lesion in monkeys would reduce the responses of thalamic neurons to graded colorectal distension (CRD) as in rats. Experiments were done on anesthetized male monkeys (Macaca fascicularis). Extracellular single cell recordings were made in the ventrolateral complex of the thalamus, mainly the ventral posterolateral (VPL) nucleus, in response to visceral and cutaneous stimulation. Of 80 VPL cells isolated, CRD activated 25, inhibited 25, and had no effect on 30 neurons. The responses of six viscerosensitive VPL neurons were recorded before and after a lesion of the DC at or above the T10 spinal segment. Lesions of other spinal tracts were made after the DC lesion. The results show that the DC lesion significantly reduced the responses of the thalamic neurons tested with CRD by >50%. Lesions of other tracts did not have a consistent effect. These results corroborate findings in the rat and support the proposal that the DC plays an important role in transmitting nociceptive visceral input into the thalamus and subsequently in visceral pain.

Sensitization of postsynaptic dorsal column neuronal responses by colon inflammation.

The role of a newly identified component of the postsynaptic dorsal column (PSDC) system in viscerosensory processing has been recently described. The purpose of this study was to examine the effect of colon inflammation on the responses of single PSDC cells, located in the vicinity of the central canal at L6-S1 spinal segments, to graded colorectal distension (CRD) and to cutaneous stimulation. Experiments were conducted on seven male Sprague-Dawley rats anesthetized with pentobarbital. Recordings were made from seven PSDC cells located around the central canal at L6-S1 in response to CRD and cutaneous stimulation before and after colon inflammation. Inflammation of the colon with mustard oil (MO) induced an increase in the background activity of these cells. Colon inflammation also potentiated the responses of the PSDC cells to graded CRD but not to cutaneous stimulation. This is consistent with previously observed effects of colon inflammation on the responses of viscerosensitive cells in the ventral posterolateral (VPL) nucleus of the thalamus and in the nucleus gracilis (NG). These observations support a role of the PSDC system in viscerosensory processing and primary visceral hyperalgesia.

Nucleus gracilis: an integrator for visceral and somatic information.

The nucleus gracilis (NG) receives an abundance of visceral input from various abdominal organs and is proposed to play an important role in visceral pain processing. The purpose of this study was to investigate the necessity of the NG for colorectal input into the ventral posterolateral (VPL) nucleus of the thalamus. Single-cell recordings were made from nine VPL cells isolated in nine different male Sprague Dawley rats anesthetized with pentobarbital sodium. Responses of the VPL cells to colorectal distension (CRD) and to cutaneous stimuli were obtained before and after lesioning of the NG. Electrolytic (n = 5) and chemical (n = 4) lesions of the NG were made in different preparations. The chemical lesions were made by injecting a solution of kainic acid into the NG. Kainic acid presumably kills neuronal cell bodies and spares axons of passage. The results indicate that a lesion of the NG, regardless of its type, reduces dramatically the responses of VPL neurons to innocuous cutaneous stimuli, and, to a lesser extent, the responses to CRD. Attenuation of VPL neuronal responses to CRD as well as to innocuous cutaneous stimuli by the NG lesions emphasizes the role of the dorsal column in visceral nociception and suggests that the NG is an integration center for visceral and cutaneous information flowing into the VPL nucleus.

Is there a pathway in the posterior funiculus that signals visceral pain?

The present report provides evidence that axons in the medial part of the posterior column at T10 convey ascending nociceptive signals from pelvic visceral organs. This evidence was obtained from human surgical case studies and histological verification of the lesion in one of these cases, along with neuroanatomical and neurophysiological findings in animal experiments. A restricted lesion in this area can virtually eliminate pelvic pain due to cancer. The results remain excellent even in cases in which somatic structures of the pelvic body wall are involved. Following this procedure, neurological testing reveals no additional neurological deficit. There is no analgesia to pinprick stimuli applied to the body surface, despite the relief of the visceral pain. Since it is reasonable to attribute the favorable results of limited midline myelotomies to the interruption of axons of visceral nociceptive projection neurons in the posterior column, we have performed experiments in rats to test this hypothesis. The results in rats indicate that the dorsal column does indeed include a nociceptive component that signals pelvic visceral pain. The pathway includes neurons of the postsynaptic dorsal column pathway at the L6-S1 segmental level, axons of these neurons in the fasciculus gracilis, and neurons of the nucleus gracilis and the ventral posterolateral nucleus of the thalamus.

Visceral nociceptive input into the ventral posterolateral nucleus of the thalamus: a new function for the dorsal column pathway.

1. Extracellular recordings were made from single neurons in the ventral posterolateral (VPL) nucleus of the thalamus in anesthetized male rats. VPL cells that responded to colorectal distension (CRD) were further tested with cutaneous and visceral stimuli before and after sequential lesions of the dorsal column (DC) and of the ventrolateral column (VLC). 2. A total of 105 VPL viscerosensitive cells was isolated. In this study, we report results from 27 cells that were tested for visceral responses before and after lesions of the DC and the VLC. 3. Ten VPL cells, isolated in 10 different rats, that responded to CRD and to activation of their cutaneous receptive fields with innocuous and noxious stimuli were tested before and after a lesion of the DC and again after a lesion of the VLC. Ten other VPL cells were isolated in 10 different rats. These were also tested for responses to CRD and to cutaneous stimuli spanning the innocuous and the noxious range before and after a lesion of the VLC and again after a lesion of the DC. 4. A lesion of the DC dramatically reduced the responses of VPL cells to CRD as well as to innocuous cutaneous stimuli. A lesion of the VLC reduced and sometimes abolished the responses of VPL cells to noxious cutaneous stimuli; however, its effect on the responses to visceral or innocuous cutaneous stimuli was minor when compared with that of a DC lesion. 5. The activity of seven VPL cells that responded to CRD and to cutaneous stimuli was observed after an injection of a chemical irritant, mustard oil (MO), into the colon. These cells showed an increase in background activity that started approximately 25-30 min after the MO injection. A lesion of the DC reduced the background activity of the cells to levels near initial values. 6. We conclude that the DC in the rat contains a pathway that is more important for transmitting visceral nociceptive signals to the VPL nucleus than are pathways in the VLC, including the spinothalamic tract. Conversely, the VLC is more important for transmitting nociceptive information of cutaneous origin.

Pelvic visceral input into the nucleus gracilis is largely mediated by the postsynaptic dorsal column pathway.

1. The purpose of this study was to investigate a proposed role for the postsynaptic dorsal column (PSDC) pathway in mediating visceral nociceptive input into the dorsal column (DC) nuclei. 2. In one group of animals, the hypogastric nerves were sectioned, thereby restricting colorectal input into the cord to pelvic afferent pathways known to coverage on lower lumbar and sacral segments. Extracellular recording were made from 41 nucleus gracilis (NG) cells that responded to colorectal distension (CRD). Results reported are from 15 NG cells that were tested before and after the administration of morphine into the sacral cord by microdialysis. 3. The responses of 11 NG cells to CRD were dramatically reduced by morphine infused into the sacral cord through a microdialysis fiber. This reduction was reversed by an intravenous injection of naloxone. Microdialysis administration of 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) or a lesion of the DC also abolished the responses of the NG cells to CRD. 4. Four NG cells that responded to CRD showed an increase in their background activity approximately 25 min after an injection of mustard oil (MO). This increase in activity was counteracted by morphine or by a lesion of the DC. 5. In a second group of animals, recordings were made from 28 PSDC cells in the L0-S1 segments of the cord. These units were antidromically activated by stimulation of the upper cervical fasciculus gracilis. The projections of five PSDC neurons into the NG were traced with the use of antidromic mapping. Results are reported for the responses of 12 PSDC cells to CRD and to cutaneous stimuli before and after morphine administration into the sacral cord by microdialysis. 6. Morphine given spinally reduced the responses of 12 PSDC cells to CRD. This reduction was reversed by an intravenous injection of naloxone. CNQX administered spinally also abolished the responses to CRD of the PSDC cells tested. 7. Four other PSDC cells were studied before and after an injection of MO into the colon. Their background activity started to increase within 25 min after the injection. Morphine suppressed this increase in background activity and this effect of morphine was reversed by naloxone. 8. The responses of NG cells to cutaneous stimuli were not significantly affected by morphine in the dose used. On the other hand, morphine significantly reduced the responses of PSDC cells to noxious cutaneous stimuli although this effect was not as dramatic as that on responses to visceral stimuli. 9. From the results of the studies described in this and the companion paper, we conclude that there is an important pelvic visceral nociceptive pathway involving PSDC neurons that synapse in the NG. The NG in turn activates neurons in the ventral posterolateral (VPL) nucleus of the thalamus. We presume that activation of VPL neurons by noxious visceral stimulation contributes to visceral pain sensation and thus that pelvic visceral pain depends largely on activity in the DC-medial lemniscus system.

Potentiation of thalamic responses to colorectal distension by visceral inflammation.

Responses of single viscerosensitive units in the ventral posterolateral (VPL) nucleus of the thalamus to cutaneous stimuli and graded colorectal distension (CRD) were recorded before and after colon inflammation by mustard oil (MO). The spontaneous firing rate increased around 25 min after the injection of MO. Responses to CRD obtained after this time increased significantly. In contrast, responses to cutaneous stimuli were attenuated and no substantial variations in the size of the cutaneous receptive fields were observed. A lesion of the dorsal column (DC) at T10 dramatically decreased the background activity and almost totally abolished the responses to CRD and to innocuous cutaneous stimuli but did not significantly affect the responses to noxious cutaneous pinch. These results indicate that colon inflammation potentiates thalamic responses to visceral stimuli while decreasing those to cutaneous stimuli.