Fos expression in spinothalamic and postsynaptic dorsal column neurons following noxious visceral and cutaneous stimuli.

The spinothalamic tract (STT) has been classically viewed as the major ascending pathway for pain transmission while the dorsal column (DC) was thought to be involved primarily in signaling innocuous stimuli. Recent clinical studies have shown that limited midline myelotomy, which transects fibers in the DC, offers good pain relief in patients with visceral cancer pain. Experimental studies provided evidence that a DC lesion decreases the activation of thalamic neurons by visceral stimuli and suggested that this effect is due to transection of the axons of postsynaptic dorsal column (PSDC) neurons. In our study, Fos protein expression in retrogradely labeled STT and PSDC neurons in the lumbosacral enlargement in rats was used as an anatomical marker of enhanced activation to compare the role of these neurons in cutaneous and visceral pain. The noxious stimuli used were intradermal injection of capsaicin and distention of the ureter. Retrogradely labeled PSDC neurons were found in laminae III-IV and in the vicinity of the central canal. STT neurons were located in laminae I, III-VII and X. Ureter distention evoked Fos expression in PSDC and STT neurons located in all laminae in which retrogradely labeled cells were found, with the maximum in the L(2) spinal segment. The Fos-positive PSDC neurons represented a significantly higher percentage of the retrogradely labeled PSDC neurons (19.3+/-2.3% SEM) than of the STT Fos-positive neurons (13.2+/-1.5% SEM). Intradermal capsaicin injection also evoked Fos expression in both PSDC and STT neurons, but with no significant difference between these two, when expressed as a percentage of the retrogradely labeled cells (11.6+/-2.9% SEM, 10.8+/-1.1% SEM). These results show that both PSDC and STT neurons are activated by cutaneous and visceral noxious stimuli. Their particular role in transmission and modulation of painful stimuli needs to be investigated further.

Postsynaptic dorsal column neurons express NK1 receptors following colon inflammation.

Recent clinical and experimental studies have suggested that the dorsal column pathway and specifically postsynaptic dorsal column neurons play an important role in the transmission of visceral pain. In our study we have mapped the distribution of postsynaptic dorsal column neurons in thoracic, lumbar and sacral spinal cord segments. The presence of immunoreactivity for neurokinin 1 receptors on these postsynaptic dorsal column neurons was examined under control conditions and after colon inflammation. The largest number of postsynaptic dorsal column neurons was found in the lumbar enlargement. They were mostly located in laminae III-IV, but depending on the spinal segment, about 7-15% of them were in the deep medial dorsal horn and in the central canal area. Under control conditions none of the 1438 postsynaptic dorsal column neurons examined expressed neurokinin 1 receptors. However, after induction of colon inflammation about 1.4% of the 2015 postsynaptic dorsal column neurons observed in the experimental group showed immunoreactivity for neurokinin 1 receptors. These neurons were preferentially found in the lower thoracic and lumbosacral spinal segments where they represented about 3-4% of the total population of postsynaptic dorsal column neurons examined. The de novo expression of neurokinin1 receptors on postsynaptic dorsal column neurons after colon inflammation suggests that substance P released from visceral primary afferents under inflammatory conditions could help produce central sensitization of these neurons.

Biological properties of copolymer of 2-hydroxyethyl methacrylate with sulfopropyl methacrylate.

Interaction of organism with non-toxic implanted polymers depends on the physicochemical properties of the implant surface, which influence the adsorption of bioactive proteins and subsequently adhesion and growth of cells. The synthetic hydrogels are known as poorly adhesive surfaces. In this study we demonstrated the adsorption of albumin, fibrinogen, fibronectin, basic fibroblast growth factor, heparin-binding epidermal growth factor-like growth factor and epidermal growth factor to poly(2-hydroxyethyl methacrylate) (pHEMA) and copolymer of 2-hydroxyethyl methacrylate (HEMA) and potassium salt of 3-sulfopropyl methacrylate (SPMAK). The adhesion and growth of 3T3 cells and human keratinocytes on surface of these polymers was tested without and with pretreatment of polymers with heparin-binding epidermal growth factor-like growth factor. The adhesion of mixture of human granulocytes and monocytes to these surfaces was also tested. The strips of both polymers were subcutaneously and intracerebrally implanted into the rat and the extent of foreign body reaction and brain biocompatibility was evaluated. The results showed the extensive adsorption of basic fibroblast growth factor and heparin-binding epidermal growth factor-like growth factor to copolymer containing SPMAK. However the adhesion (and growth) of cells to this type of copolymers was very low. Preadsorption of human plasma to pHEMA clearly stimulated the leukocyte adhesion in contrary to copolymer containing SPMAK. The extent of foreign-body reaction was significantly higher against the pHEMA compared to tested copolymer p(HEMA-co-SPMAK). In conclusion, the tested copolymer was a poorly adhesive substrate that is only poorly recognized by the non-specific immunity, although the adsorption of basic growth factors to this substrate is highly significant. Both polymers were well tolerated by the brain tissue. The phenotype of surrounding neurons was more close to the control neurons in the brain tissue surrounding the p(HEMA-co-SPMAK) implants.

Biocompatibility of HEMA copolymers designed for treatment of CNS diseases with polymer-encapsulated cells.

Surrounding the cells with a semipermeable polymeric membrane allows transplanting unmatched xenogeneic cells without a risk of their rejection. We prepared and tested several 2-hydroxyethyl methacrylate (HEMA) copolymers with alkyl methacrylates or acrylates to find out which was the most valuable for cell encapsulation. On the basis of optimum physical properties and good results of cytotoxicity tests, HEMA-EMA copolymer was chosen as a suitable candidate for encapsulation and immunoprotection of xenogeneic cells before their grafting into the central nervous system (CNS). To characterize the biocompatibility of p(HEMA-co-EMA) copolymer in the CNS, we implanted microcapsules made of this hydrogel into the brains of adult rats that were allowed to survive for 0.5, 1, 3, 6, and 9 months. Analysis of histological sections containing the implantation site was aimed at assessment of the cellular density at the implant-brain interface and identification of cell types participating in a tissue reaction. Our results indicated that the tissue reaction that was observed was caused largely by the implantation procedure because HLA-DR- and GSI-B4-positive macrophages/microglia infiltrated mainly the implantation channel. The number of these cells declined with time, which was true also for GFAP-positive reactive astrocytes, as well as for foreign body giant cells. The amount of connective tissue components surrounding the implanted microcapsules increased only slightly. These findings indicated that p(HEMA-co-EMA) hydrogel was well tolerated after implantation in the brain.

Nitric oxide mediates the central sensitization of primate spinothalamic tract neurons.

Nitric oxide (NO) has been proposed to contribute to the development of hyperalgesia by activating the NO/guanosine 3',5'-cyclic monophosphate (cGMP) signal transduction pathway in the spinal cord. We have examined the effects of NO on the responses of primate spinothalamic tract (STT) neurons to peripheral cutaneous stimuli and on the sensitization of STT cells following intradermal injection of capsaicin. The NO level within the spinal dorsal horn was increased by microdialysis of a NO donor, 3-morpholinosydnonimine (SIN-1). SIN-1 enhanced the responses of STT cells to both weak and strong mechanical stimulation of the skin. This effect was preferentially on deep wide dynamic range STT neurons. The responses of none of the neurons tested to noxious heat stimuli were significantly changed when SIN-1 was administered. Intradermal injection of capsaicin increased dramatically the content of NO metabolites, NO-2/NO-3, within the dorsal horn. This effect was attenuated by pretreatment of the spinal cord with a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). Sensitization of STT cells induced by intradermal injection of capsaicin was also prevented by pretreatment of the dorsal horn with the NOS inhibitors, L-NAME or 7-nitroindazole. Blockade of NOS did not significantly affect the responses of STT cells to peripheral stimulation in the absence of capsaicin injection. The data suggest that NO contributes to the development and maintenance of central sensitization of STT cells and the resultant mechanical hyperalgesia and allodynia after peripheral tissue damage or inflammation. NO seems to play little role in signaling peripheral stimuli under physiological conditions.

Effect of chemical structure of hydrogels on the adhesion and phenotypic characteristics of human monocytes such as expression of galectins and other carbohydrate-binding sites.

The reactivity of diverse immune aspects to the presence of synthetic polymers represents one of the most important aspects of implantable device biocompatibility. In this report, we show the effect of the chemical structure of a synthetic polymer support on monocyte adhesion and selected phenotypic characteristics in vitro as a model for the initial steps of non-self-recognition of an implant. The extent of monocyte adhesion was significantly influenced by the support chemistry. The highest level of monocyte adhesion was observed on a surface copolymer of 2-hydroxyethyl methacrylate with dimethyl aminoethyl methacrylate relative to results of experiments in which poly(2-hydroxyethyl methacrylate) or the copolymer of 2-hydroxyethyl methacrylate with the sodium salt of methacrylic acid was used. Cell adhesion to the polymers tested and to glass was accompanied by enhanced expression of the carbohydrate-binding sites tested for asialoglycoprotein beta-galactosides such as galectins, beta-N-acetylgalactosamine, alpha-mannoside, specific lectin for heparin as well as the lymphokine-macrophage migration inhibitory factor in the monocytes tested. These results suggest the importance of monocyte adhesion to the biomaterial surface for their development into macrophages and further non-self-recognition of the implanted device.

Neurokinin 1 and 2 antagonists attenuate the responses and NK1 antagonists prevent the sensitization of primate spinothalamic tract neurons after intradermal capsaicin.

1. Activation of neurokinin receptors contributes to the excitation of many dorsal horn neurons by cutaneous sensory stimuli, particularly noxious stimuli. In the present study we investigate the role of neurokinin receptors in the activation of primate spinothalamic tract (STT) neurons by cutaneous mechanical stimuli and by intradermal injection of capsaicin. This was done by testing the responses of these neurons to a battery of cutaneous stimuli before and during infusion by microdialysis of antagonists selective for NK1 and NK2 receptors. 2. The NK1 receptor antagonists cis-3-(2-methoxybenzyl-amino-2-benzhydrylquinuclidine (CP96345) and D-Pro9-[Spiro-y-lactam]-Leu10,Trp11)-Physalaemin(1-11) (GR82334) did not significantly reduce the responses of STT cells to mechanical stimulation of the skin. Both NK1 antagonists did, however, produce a significant reduction in the responses of STT neurons to an intradermal injection of capsaicin. Finally, despite having no effects on responses to mechanical stimuli, both NK1 antagonists prevented the sensitization of the responses to cutaneous stimuli that is usually observed after intradermal injections of capsaicin. 3. The NK2 selective antagonists PhCO-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (GR98400) and [Tyr5,D-Trp6,8,9,Lys10]-NKA (4-10) (MEN10376) had effects very similar to those of the NK1 antagonists, but with an important difference. Neither NK2 antagonist affected the responses of STT neurons to noxious or innocuous mechanical stimulation of the skin, but they did reduce the responses to intradermal capsaicin injections. These compounds failed to prevent capsaicin-induced sensitization. In fact, cells exposed to GR98400 or MEN10376 showed unusually sustained increases in the responses to mechanical stimuli after the first capsaicin injection, suggesting that these compounds actually induced sensitization. 4. These results support the contention that both neurokinin receptors participate in the processing of nociceptive information in the dorsal horn, especially responses to strong stimuli such as intradermal injection of capsaicin. NK1 receptors are also involved in the sensitization of STT neurons after peripheral injury. A clearer understanding of the role of NK2 receptors in sensitization requires further studies with improved antagonists.

The effect of trans-ACPD, a metabotropic excitatory amino acid receptor agonist, on the responses of primate spinothalamic tract neurons.

The responses of primate spinothalamic tract (STT) neurons to innocuous and noxious mechanical stimuli applied to the skin can be enhanced for more than an hour following prolonged noxious stimulation. This increased responsiveness is thought to reflect sensitization of dorsal horn neurons and may help account for secondary hyperalgesia and mechanical allodynia. The proposal that central sensitization is due to the activation of second messenger system was tested in this study by examining the effect of trans-ACPD (trans-D,L-1-amino-1,3-cyclopentanedicarboxylic acid), an agonist of metabotropic excitatory amino acid (EAA) receptors, introduced into the dorsal horn by microdialysis. A low dose of trans-ACPD resulted in an increase in the responses of STT cells to an innocuous mechanical stimulus (BRUSH), but no increase in the responses to noxious mechanical and thermal stimuli or in the excitation produced by iontophoretically applied EAAs. A high dose of trans-ACPD caused a transient increase in background activity, but no change in the responsiveness of spinothalamic cells to any of the test stimuli. It is concluded that low doses of trans-ACPD can selectively enhance transmission through interneuronal pathways mediating tactile inputs to spinothalamic cells.

The effect of phorbol esters on the responses of primate spinothalamic neurons to mechanical and thermal stimuli.

1. Sensitization of dorsal horn neurons is thought to play an important role in pain perception, secondary hyperalgesia, and allodynia. Recent experimental evidence suggests that the sensitization of dorsal horn neurons is induced by combined increased release of excitatory amino acids and peptides in the spinal cord dorsal horn from nociceptive primary afferents due to an injury-caused barrage of impulses. We tested the hypothesis that protein kinase C (PKC) is involved as a second messenger in this process of neuronal sensitization. To activate PKC, infusion of a phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] into the dorsal horn through a microdialysis fiber was used. During TPA infusion the background activity of spinothalamic (STT) neurons increased substantially. After TPA application, while the background activity of the STT neurons was still increased, the responses evoked by either innocuous or noxious mechanical stimulation of the cutaneous receptive field did not change from the control level. However, 1 h after TPA administration the background activity returned to the control level and responses to innocuous mechanical stimuli were significantly elevated. The responses of STT cells to noxious heat and noxious mechanical stimuli did not change significantly after TPA administration. When a phorbol ester that does not activate PKC was applied (alpha-TPA), no significant changes in background or evoked activity of STT cells were observed. Our results provide evidence that PKC may play an important role in the process of sensitization of dorsal horn neurons to innocuous mechanical stimuli.

Responses of spinothalamic tract neurons to mechanical and thermal stimuli in an experimental model of peripheral neuropathy in primates.

1. An experimental peripheral neuropathy (EPN) was induced in three monkeys (Macaca fascicularis) by ligation of spinal nerve L7. Behavioral responses to innocuous mechanical stimuli were tested before and after the surgery. Two weeks after the nerve ligation, the activity of spinothalamic tract (STT) neurons was recorded on both sides of the spinal cord with the animal under general anesthesia. Responses of the STT neurons to the following stimuli applied to the skin were recorded: graded mechanical stimuli (brush, press, pinch and squeeze), von Frey filaments of different bending forces (0.077-19.05 g), 5-s heat stimuli ranging from 39 to 53 degrees C, and 15 s cold stimuli (32-8 degrees C). 2. Innocuous mechanical stimulation of the foot did not evoke hindlimb withdrawal in the animals before surgery. Within 24-48 h after nerve ligation, the animals showed hindlimb withdrawal to the same innocuous stimuli. This behavior was more pronounced on the side of the ligation than on the sham-operated side and more frequent during the second week after the surgery. 3. Responses of 51 STT neurons recorded on the side of the ligation (EPN all group) were compared with responses of 33 STT cells recorded on the sham-operated side (control group) and with records from STT neurons in unoperated animals obtained earlier (reference group). Neurons from the EPN all group were divided into two sets according to their rostrocaudal location (EPN R, rostral to L6/7 border, n = 40; EPN C, caudal to L6/7 border, n = 11). 4. Neurons from the EPN all and EPN R groups had significantly higher background frequencies than those from the control and reference groups. Innocuous brush stimuli evoked mean discharge frequencies of approximately 35 Hz in EPN R neurons and only approximately 15 Hz in both control and reference groups. Increased responsiveness of EPN R neurons to innocuous stimuli was also demonstrated by lower thresholds and higher discharge frequencies to von Frey filament stimulation and by discriminative analysis of the responses evoked by graded mechanical stimuli. 5. The responses of the EPN R neurons to heat stimulation of the skin showed decreased thresholds and increased responses to suprathreshold stimuli, resulting in a significant leftward shift of the stimulus-response curve compared with both reference and control groups. The neurons from the control group showed responses comparable to reference group values. 6. Neurons from the reference group tested with the cooling stimuli showed no evoked response above background.(ABSTRACT TRUNCATED AT 400 WORDS)

The non-NMDA antagonist CNQX prevents release of amino acids into the rat spinal cord dorsal horn evoked by sciatic nerve stimulation.

Basal extracellular concentrations of 9 amino acids (AAs: aspartate, Asp; glutamate, Glu; asparagine, Asn; serine, Ser; glycine, Gly; threonine, Thr; alanine, Ala; taurine, Tau; and glutamine, Gln) were determined in the spinal cord dorsal horn of anesthetized rats using microdialysis and HPLC techniques. The concentrations of all measured AAs but Gln increased significantly (P < 0.05) during sciatic nerve stimulation at C-fiber strength. The concentration of Tau remained elevated following stimulation, while the other AAs returned to prestimulation values. Addition of the specific non-NMDA antagonist, CNQX, to the perfusing solution prevented the nerve stimulation-evoked AA release. Since the measured increases in extracellular AA concentrations are probably mainly due to activation of interneurons, these results suggest that blockade of non-NMDA receptors prevented activation of interneurons in the dorsal horn and support a major role of non-NMDA receptors at the first synapse of primary afferent fibers in the dorsal horn. Complete block of AA release and decreased basal levels of Glu after infusion of TTX into the dorsal horn also implies increased neuronal activity as the main source of higher AA levels during nerve stimulation.

The role of NMDA and non-NMDA excitatory amino acid receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal, and electrical stimuli.

The role of excitatory amino acids (EAAs) in the excitation of monkey spinothalamic tract (STT) neurons following activation of cutaneous primary afferent fibers by noxious and non-noxious stimuli was investigated. The responses of STT neurons to either NMDA or non-NMDA EAA ligands were blocked by infusion of specific antagonists through a microdialysis fiber into the region surrounding the cells. Our results show that blockade of non-NMDA receptors results in a nearly complete elimination of the responses of STT neurons to all stimuli. Blockade of NMDA receptors results in an attenuation of the responses to noxious stimuli but, in addition, prevents the development of the sensitization of STT neurons that is often observed after intradermal injection of capsaicin. These observations further support a role of EAAs in the transmission of sensory information from primary afferent fibers to dorsal horn neurons and a role for NMDA receptors in the generation of hyperalgesia.

Responses of spinothalamic tract cells to mechanical and thermal stimulation of skin in rats with experimental peripheral neuropathy.

1. Responses of spinothalamic tract (STT) neurons to mechanical and thermal stimulation of skin were recorded under urethane and pentobarbital anesthesia in 12 control rats and in 20 rats with experimental neuropathy. Activity of the STT cells in neuropathic rats was recorded 7, 14, and 28 days after inducing the neuropathy by placing four loose ligatures on the sciatic nerve. 2. All neuropathic animals showed guarding of the injured hindpaw and a shorter withdrawal latency from a radiant heat source of the neuropathic hindpaw than that of the sham-operated paw. 3. STT neurons in neuropathic animals showed the most profound changes 7 and 14 days after the nerve ligation. When compared with STT cells in unoperated animals, approximately half of the neurons had high background activity, responses to innocuous stimuli represented a larger percentage of the total evoked activity in wide dynamic range neurons, and the occurrence and magnitude of afterdischarges to mechanical and thermal stimuli were increased. 4. The mean threshold temperatures of heat-evoked responses of the STT cells in neuropathic animals were not different than those of cells from control animals. However, in neuropathic rats, cells reacting to small heat stimuli usually already had afterdischarges. 5. The increase in the background activity of STT cells is consistent with behavioral observations of spontaneous pain in this model of experimental neuropathy. Furthermore, the afterdischarges of STT cells may parallel the prolonged paw withdrawal in response to noxious stimuli that is seen in these animals and that is evidence for hyperalgesia. However, there was no indication of a lowered threshold for thermal stimuli as might be expected if the animals have thermal allodynia. Mechanical allodynia may have resulted from a relative increase in responsiveness to innocuous mechanical stimuli. However, responses to noxious mechanical stimuli were reduced compared with control, at least at 28 days after the ligation. Peripheral and central mechanisms responsible for the changes in responses of STT cells in neuropathic animals are suggested.