Unilateral spinal nerve ligation leads to an asymmetrical... : PAIN (original) (raw)
1. Introduction
Mast cells are found in the brain of normal mammals, including humans (Dropp, 1972, 1976; Theoharides, 1990). They are predominantly located in the thalamus in rats (Florenzano and Bentivoglio, 2000; Goldschmidt et al., 1985) and mice (Yang et al., 1999), closely apposed to neurons (Dines and Powell, 1997; Manning et al., 1994) and on the parenchymal side of blood vessels (Dimitriadou et al., 1990; Manning et al., 1994; Yang et al., 1999). Mast cell populations in the brain are influenced by a variety of factors, including stress (Cirulli et al., 1998; Theoharides et al., 1995) and reproductive hormones (Asarian et al., 2002; Wilhelm et al., 2000; Zhuang et al., 1993, 1997). While the thalamus is important in the integration of sensory and motor information, the function of these cells within the thalamus is not known.
Mast cells synthesize and release several substances relevant to nociception, including neurotrophins, such as nerve growth factor (NGF) (Leon et al., 1994; Skaper et al., 2001), cytokines such as tumor necrosis factor alpha (TNF-α) (Cocchiara et al., 1999; Gordon and Galli, 1990; Skaper et al., 2001), interleukins (Galli, 1993) and histamine (Barke and Hough, 1993; Goldschmidt et al., 1985). Substances released from nerves, such as NGF and substance P, can activate mast cells in skin (Foreman, 1987) and brain (Cocchiara et al., 1999; Dimitriadou et al., 1990; Ottosson and Edvinsson, 1997; Rozniecki et al., 1999). Cytokines, neurotrophins, serotonin (5-HT) and histamine from mast cells (Lambracht-Hall et al., 1990; Theoharides et al., 1985; Vliagoftis et al., 1990), in turn, sensitize nociceptors (Mendell et al., 1999; Nakano and Taira, 1976; Taiwo and Levine, 1992) and increase their rate of firing. Their juxtaposition to sensory neurons, particularly substance P-containing primary afferent C-fibers in the periphery (Dines and Powell, 1997; Oura et al., 1992) and dura (Dines and Powell, 1997; Keller and Marfurt, 1991; Lambracht-Hall et al., 1990; Rozniecki et al., 1999) is consistent with a pronociceptive role in the periphery.
While several studies support a role for mast cells in the development of hyperalgesia in the periphery (Lewin et al., 1994; Zuo et al., 2003), the response of mast cells in the brain to noxious stimuli has not been studied. To address this issue, we examined whether the mechanical hyperalgesia induced by spinal nerve (L5) ligation (SNL) changes either the population of thalamic mast cells or their state of granulation. We hypothesized that mast cells in the thalamus would respond to nociceptive signaling by degranulating in response to the massive input, along substance P-containing spinothalamic tract neurons (Battaglia and Rustioni, 1992; Battaglia et al., 1992; Nishiyama et al., 1995). Because SNL produces a unilateral nociceptive input, we further hypothesized that mast cells on each side of the thalamus would be differentially affected by SNL. In contrast, a bilateral effect would be predicted if noxious stimuli, that are known to activate the hypothalamic-pituitary-adrenal (HPA) axis (Aloisi et al., 1995; Culman et al., 1997), were responsible for changes in mast cells. Females are generally more sensitive to nociceptive stimuli than males in animal models of neuropathic (Coyle et al., 1995, 1996; Lavand'homme and Eisenach, 1999) and inflammatory pain (Tall and Crisp, 2004). Because of these gender differences in the perception of pain as well as the sensitivity of mast cells to reproductive hormones, we compared responses of mast cells to nociceptive input in females to that in males.
2. Materials and methods
2.1. Animals
Adult Swiss-Webster mice of both sexes weighing 20–30g (Harlan, Indianapolis, IN) were used. Animals were housed separately by sex, five females or four males per cage, and allowed to acclimate for at least 1 week prior to use. Mice were allowed free access to food and water, and housed in a room with a constant temperature of 23°C on a 12-h light:12-h dark cycle. Animals were used strictly in accordance with the Guidelines of IASP and the University of Minnesota Animal Care and Use Committee, and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHEW Publication (NIH) 78-23, revised 1995).
2.2. Drugs and chemicals
All chemicals, including sodium pentobarbital and salts for preparing phosphate buffered saline (PBS) were purchased from Sigma Chemical Company (St Louis, MO).
2.3. Ligation of L5 spinal nerve (SNL)
The method of SNL described by Kim and Chung (1992) in rats and by Fairbanks et al. (2000) in mice was followed. Mice were anesthetized with sodium pentobarbital (5mg/kg, i.p.; supplemented as necessary), and placed in a prone position. Using aseptic conditions, the left paraspinal muscle was separated from the spinous processes at the L4–S2 levels and removed to permit visualization of the L6 transverse process and the rostral tip of the sacrum. The L6 transverse process was carefully removed using a fine forcep (0.3×0.25mm) (Fine Science Tools No. 00108–11, Foster City, CA) to permit visual identification of the L4–L5 spinal nerves. The L5 spinal nerve was then tightly ligated with 6–0 silk suture distal to the dorsal root and proximal to the confluence of spinal nerves L4, L5 and L6. The incisions were closed and secured by the placement of stainless steel wound clips. Identical operation and handling, but without nerve-ligation, was performed on sham control mice. To assess the effect of denervation, an additional group was operated, as described, but the nerve was transected.
2.4. Histological preparation of tissue
Mice were anesthetized intraperitoneally (i.p.) with sodium pentobarbital (60mg/kg) prior to transcardial perfusion with 15ml of ice-cold phosphate-buffered saline (PBS, pH 7.4) followed by 35ml of ice-cold PBS containing 4% formaldehyde (pH 6.9). After perfusion, brains were rapidly removed and stored in the same fixative solution overnight and thereafter in PBS solution containing 30% sucrose for an additional 2 days. Using a freezing, sliding microtome, brains were sectioned at 40-μ in the coronal plane, slices were mounted on gelatin-coated slides and stained with 0.125% acidified (pH 2–2.5) aqueous toluidine blue (J.T. Baker Chemical Co., Phillipsburg, NJ) solution for 30min (Florenzano and Bentivoglio, 2000). Toluidine blue is an aniline dye that binds to sulfated glycosaminoglycans of the mast cell granules. Sections were dehydrated in increasing alcohol series, dipped in xylene and coverslipped with DPX solidifying mountant for light microscopic histology. Light microscopy identified mast cells by their metachromatic purple cytoplasmatic granules while the neurons and surrounding tissue stained light blue. Mast cells are usually found scattered, often in clusters, throughout the thalamus. Because of this, systematic sampling or random analysis of sections may yield artificially high or low numbers depending on the location of selected sections relative to these pockets of mast cells. For this reason, we evaluated the number and distribution of mast cells throughout the entire anterior to posterior extent of the thalamus, noting their relative state of granulation and nuclear location, as described previously (Taiwo et al., 2004).
2.5. Measurement of granulation state
Because the degree of degranulation has been taken to suggest the degree of activity of these cells, we established an arbitrary scoring system to differentiate between the extreme stages of granulation found amongst cells visualized using toluidine blue. Mast cells exist in either fully granulated (darkly metachromatically stained) or faintly granulated (pinker and pale) states. Mast cells were classified as degranulated if the structure of the cell membrane was not intact or the metachromatic stain was relatively more pink and less intense compared to other mast cells in the same thalamus. The majority of mast cells in a normal, untreated mouse were fully granulated (dark blue or black throughout the cytosol) while only a minority were considered to be partially degranulated based on their relatively more pinkish and pale contents surrounding a visible nucleus. A high degree of correspondence (greater than 95%) between two independent observers is found using this approach (Taiwo et al., 2004).
2.6. Analysis of data
Thalamic mast cell numbers and their state of degranulation were monitored as described above. Mast cells with nuclei in the focal plane were counted. Because the number of mast cells in the rodent thalamus show noticeable variability and are distributed in clusters in close proximity to the vasculature, serial reconstruction of the whole thalamic area was required (about 90 slides/animal) (Coggeshall and Lekan, 1996; Guillery and Herrup, 1997). The diencephalon from the anterior comissure to the anterior midbrain was cut and mapped based on the Mouse Brain Atlas by Paxinos and Franklin (2001). Prior to statistical analysis, the Abercrombie correction factor is applied to minimize the possibility of double counting of cells in consecutive tissue sections (Abercrombie, 1946). Data, reported as the mean±SEM, were analyzed and significance determined using either Student's _t_-test (between two groups) or ANOVA followed by Fisher Protected Least Squares Difference (PLSD) test, as appropriate.
3. Results
3.1. Behavioral analysis
To quantify the development of mechanical hyperalgesia at various times after surgery, male and female mice were tested by measuring the number of behavioral withdrawal responses to a #3.84 von Frey fiber (0.6g delivered 10 times on the plantar surface of each paw) after SNL and sham-operation. Separate groups of mice were tested at each time-interval immediately prior to death on day 2, 7 or 14. Unilateral ligation of the L5 spinal nerve was successful in producing hyperalgesia as the hind paw was withdrawn more frequently on the side ipsilateral to the SNL surgery than the side contralateral to L5 nerve injury (Fig. 1). When data from male and female mice were pooled, differences in ipsilateral compared to contralateral responses were statistically significant at both 2 and 7 days with recovery by day 14 after SNL. When these same data were analyzed separately, the sensitivity of male and female mice to SNL was similar in magnitude. However, the average number of responses to von Frey fiber stimulation was no longer statistically different between ipsilateral and contralateral paws in males at day 7 while these values remained significant when analyzed in females. No differences were seen in mechanical sensitivity between the ipsilateral and contralateral sides in sham-operated female or male mice at any time measured.
Time-course of mechanical hyperalgesia in male and female mice. The mean (±SEM) number of paw withdrawal responses to probing with a #3.84 von Frey fiber (0.6 g) out of a possible total of 10 on each side are shown for L5 spinal nerve-ligated (SNL) and sham-operated (Sham) controls. Asterisks indicate a statistically significant difference in the number of paw-withdrawal responses between the ipsilateral and contralateral paws when analyzed by Student's _t_-test with a cutoff of P<0.05. The number of mice in each group is shown at the base of each pair of bars throughout the figures.
Because it is unclear whether SNL surgery influences the contralateral paw, we also analyzed our data by comparing responses of the ipsilateral paw in mice after SNL to that in mice after sham-surgery. In spite of the variability in von Frey fiber responses in different groups of mice, when responses of the ipsilateral paw of males and females (pooled) were compared between SNL and sham-surgery, values differed significantly at day 7. When separated by sex, only females still retained a significantly greater number of responses at day 7 while males did not. This alternate method of analysis again supports the efficacy of the surgery and a slightly greater sensitivity of females than males to SNL.
3.2. Number of mast cells in the whole thalamus
Mice were killed at 2, 7 or 14 days after surgery and the brains analyzed. The total number of mast cells and the number that were degranulated in either SNL or sham-operated mice were generally comparable to those in un-operated, naïve, historical control groups of the same sex, weight and strain (Fig. 2). Similarly, the total numbers and the numbers degranulated in nerve-ligated mice were no different than in sham-operated mice of either gender on the days examined.
Number of mast cells in the thalamus of male and female mice. The mean (±SEM) number of mast cells in the whole thalamus (top panel) and the number of those that are degranulated (bottom panel) after L5 spinal nerve-ligation (SNL) or sham-operation (Sham) were generally comparable to the values previously observed in historical control mice of each sex. When analyzed using Student's _t_-test (P<0.05), no statistically significant differences were seen between SNL mice and sham-operated controls in the total number of mast cells (top panel) or the number that were degranulated (bottom panel) at each time indicated.
3.3. Thalamic mast cells in ipsilateral and contralateral hemispheres
Spinothalamic input from the body generally crosses to innervate areas of the contralateral thalamic nuclei involved in sensory-discriminative aspects and location of painful stimuli. Based on this, we examined the relative distribution of mast cells in each hemisphere following nociceptive input induced by SNL. When analyzed by absolute number, the population of mast cells on the contralateral side of the surgery did not differ from that on the ipsilateral side after either SNL or the sham procedure. However, the variability in the absolute number of thalamic mast cells in one animal compared to another precluded an accurate assessment of the effect of ligation due to the large standard errors. To standardize these values, we determined the percent of the total number of mast cells in each mouse that were located on each side of the thalamus, and expressed those values as a mean, as shown in Fig. 3. In addition, we calculated the percent of the total mast cell population in each animal that was degranulated (Fig. 3, lower panels). Using this approach, the mast cell population in female mice on the side of the thalamus contralateral to the nerve-ligation was more than double that on the side contralateral to the injury on days 2 (73.9±13.8% vs. 26.1±13%) and 7 (77.7±5.9% vs. 24.9±6.4%). These times correspond to those when females were found to be hyperalgesic. Similarly, the percentage of mast cells that were degranulated was greater on the contralateral side than on the ipsilateral side on days 2 (19.9±11.3% vs. 0.8±0.6%) and 7 (15.9±3.8% vs. 5.3±3.5%). These relative differences in distribution were no longer observed on day 14, coincident with the recovery of females from the hyperalgesic effect of SNL. In contrast, the percent of the total number of mast cells and the extent of their degranulation on either side of the thalamus did not differ in either sham-operated females or males following either surgical procedure. In addition, mast cells in females whose spinal nerves were transected completely rather than ligated were symmetrically distributed (44.5±21.3% compared to 55.5±21.3%, _n_=5), similar to those in sham operated females.
Relative distribution of mast cells on the ipsilateral and contralateral sides of the thalamus. The mean (±SEM) percent of mast cells (top panel) and the percent that were degranulated (bottom panel) on the side of the thalamus ipsilateral and contralateral to L5 spinal nerve-ligation (SNL) or sham-surgery (Sham) are depicted in female and male mice. These values are derived from the same data as shown in Fig. 3, but expressed as the mean percent of the total population of mast cells in each mouse. This manipulation standardizes the census from one mouse to another and decreases the variability. Asterisks indicate a significant difference in the percent of mast cells when comparing the ipsilateral to the contralateral sides using Student's _t_-test at a cutoff of P<0.05.
3.4. Rostral-caudal distribution of mast cells in female mice
Because of the relatively greater distribution of mast cells on the side contralateral to nerve-ligation on days 2 and 7 in female mice, we questioned whether there was a differential distribution of these cells to specific areas of the thalamus. To identify clusters in their general rostral-caudal distribution within the two hemispheres, we compared their relative numbers, expressed as a percent of the total in each animal, on each side of the ninety 40-μ sections of nerve-ligated and sham-operated females (Fig. 4A). Data from days 2 and 7 were pooled. The relative distribution of mast cells on the contralateral side of nerve-ligated females was found to be greater than on the ipsilateral side in sections 35, 36, 37, 42 and 43. The percent that were degranulated was also greater in this region, suggesting that these cells are actively degranulating. This analytical approach did not reveal any difference in the distribution of mast cells in sham-operated females (Fig. 4B).
Relative rostral-caudal distribution of thalamic mast cells in each hemisphere of female mice. Mast cells in each section are expressed as the mean (±SEM) percent of the total population of mast cells in each female mouse (A) after L5 spinal nerve-ligation (SNL) or (B) sham-surgery. Data from mice 2 and 7 days after surgery were pooled at both times to reflect intervals during which females were hyperalgesic. Asterisks indicate that the value ipsilaterally differs from that found contraterally in each section using Student's _t_-test and a cutoff of P<0.05.
3.5. Distribution of mast cells within thalamic nuclei
The distribution of mast cells within sections 35, 36, 37, 42 and 43 was surveyed for their exact location. Because female mice were hyperalgesic on days 2 and 7 after SNL, groups from these two days were pooled creating a population of 19 females that were nerve-ligated and 10 that were sham-operated. We then drew a composite of their cumulative population densities in these sections on the appropriate representation from a mouse atlas at that location (Bregma −1.40 to 1.82), as shown in Fig. 5. These pictures illustrate that the composite of mast cells on the contralateral side were notably more dense, especially in regions of the ventral posteromedial (VPM) and posterior (Po) nuclei in SNL mice. In contrast, mast cells were more evenly distributed on both sides of sham-operated mice.
Distribution of mast cells located in thalamic sections 35, 36, 37, 42 and 43 in L5 nerve-ligated (SNL, top) and sham-operated (bottom) female mice. Filled black circles (•) represent granulated mast cells while open circles (○) represent mast cells with some degree of degranulation. The composites represent the distribution of all mast cells found in these sections of all 19 spinal nerve-ligated and all 10 sham-operated control mice evaluated in Figs. 3 and 4 where days 2 and 7 are pooled. This analysis illustrates the asymmetrical distribution of mast cells in SNL mice, such that mast cells are more numerous on the contralateral side, whereas in sham-operated controls, they are more evenly distributed.
When analyzed throughout the entire rostral-caudal extent of each nucleus on days 2 and 7, mast cells in the contralateral nuclei were generally more numerous relative to those on the ipsilateral side in mice after SNL. In particular, populations in the Po and lateral geniculate (LG) on the side contralateral to SNL were at least twice the population of those on the ipsilateral side (Fig. 6A). While these changes are statistically significant, they are small. The mean values that these percentages reflect (Po: 5.7±1.9 contralateral and 3.1±2.1 ipsilateral; LG: 3.8±1.0 contralateral and 1.1±0.6 ipsilateral) are low and contribute only minimally to the large difference between thalamic hemispheres. In sham-operated mice, mast cells were otherwise variably distributed in both ipsilateral and contralateral thalamic nuclei, with no difference in nuclear or hemispheric distribution.
Relative distribution of mast cells in thalamic nuclei. The mean percent (±SEM) of the total population of mast cells (A) and the percent that are degranulated (B) in each thalamic nucleus are shown in female mice on days 2 and 7. The asterisk indicates the nuclei (Po and LG) where the percent of mast cells is greater on the contralateral side than on the ipsilateral side in spinal nerve-ligated (SNL) mice when analyzed using Student's _t_-test at a cutoff of P<0.05. Thalamic nuclei are abbreviated as follows: PV, paraventricular; Re, reuniens; Rh, rhomboid; Rt, reticular; CL, centrolateral; CM, central medial; PC, paracentral; LD, laterodorsal; LP, lateral posterior; VL, ventrolateral; VM, ventromedial; Gus/visc, gustatory/visceral; VPL, ventral posterolateral; VPM, ventral posteromedial; Sub, submedius; SM, stria medularis; Po, posterior; LG, lateral geniculate; MG, medial geniculate; and PF, parafascicular, as taken from Paxinos and Franklin (2001).
Finally, we compared the relative distribution of mast cells located bilaterally in each nucleus of our naïve historical control females to that in the present experiments. Only one nucleus, the parafascicular (PF), differed such that the percent of the total population of mast cells after SNL (0.4±0.4%) was significantly less than in either historical control (7.17±2.47%) or sham-operated (13.9±9.4%) females (ANOVA, PLSD; P<0.05). By day 14, this difference was no longer present. In contrast, the mast cell population in the PF of male mice on day 2 after SNL, a time when males were measurably hyperalgesic, did not differ from that in either historical control or sham-operated male mice.
4. Discussion
Using a model of chronic pain involving injury to the fifth lumbar spinal nerve, we investigated the influence of SNL on the number, distribution and state of granulation of thalamic mast cells. The major findings of this study are that thalamic mast cells in mice are sexually dimorphic in their response to nerve injury-induced mechanical hyperalgesia. Specifically, mast cells are increased on the side of the thalamus receiving nociceptive input from the injured nerve in female but not male mice. The resulting asymmetry supports the hypothesis that nociceptive input to the thalamus, rather than stress hormones released in response to noxious stimulation, is responsible for these effects. The relatively greater impact of SNL in females on both the expression of hyperalgesia as well as the distribution of thalamic mast cells is consistent with a possible association between thalamic mast cells and nociception.
The mechanical hyperalgesic effect of this model was validated by increased hind paw behavioral responses after SNL and the absence of hyperalgesia in sham-operated mice. The duration of hyperalgesia in nerve-ligated females and males differed by sex, consistent with previous studies. The more persistent sensitivity of females (days 2 and 7) than males (day 2) to nerve-ligation is in keeping with the tendency for females to be more sensitive to nociception (Coyle et al., 1996; Fillingim and Maixner, 1995; Frot et al., 2004; Sarlani et al., 2004; Wise et al., 2002). This sex difference likely results from a gonadal influence as intact male rats are less susceptible to a repetitive nociceptive stimulation than gonadectomized male mice (Aloisi et al., 2003) while intact female rats are more sensitive to tactile stimuli than ovariectomized rats (Coyle et al., 1996). Thus, sex hormones are important modulators of nociceptive processing.
When mast cells were examined, the variability in their numbers from one mouse to another precluded a simple analysis of their absolute populations. However, when their numbers were standardized, by expressing them as a percent of the total population in each mouse, a pattern emerged. The results of these analyses are consistent with a sexually dimorphic and unilateral influence of SNL on thalamic mast cells. Specifically, the percent of mast cells on the side of the thalamus contralateral to SNL was greater than the number on the ipsilateral side. This occurred only on days 2 and 7 and only in female mice contemporaneously with the manifestation of hyperalgesia. This suggests that sensory changes induced by SNL are associated with the genesis of this unique profile of mast cells. Although an asymmetric distribution of brain mast cells has been reported in naïve female rats (Goldschmidt et al., 1984), sheep, hedgehog and dog (Michaloudi and Papadopoulos, 1999), we did not detect lateralization of mast cells in mice after sham-operation, nerve-transection or after the effect of SNL had disappeared (day 14). In addition, the side with more mast cells in SNL females was consistently the right hemisphere while that in females of previous studies was consistently the left hemisphere. The asymmetric distribution of mast cells after SNL was accompanied by a corresponding increase in their incidence of degranulation, suggesting that this enlarged pool is active. These differences in distribution and degranulation were not observed in sham-operated females and are no longer seen on day 14 after SNL, a time when females had recovered from hyperalgesia. Together these data support a relationship between mast cells and hyperalgesia.
When this method of analysis was applied to males, no differences were detected. The failure to detect an asymmetrical distribution of mast cells in males during their hyperalgesic phase on day 2 may result from a poor signal to noise ratio or the different effects of estrogen and testosterone on mast cells and nociception. Hormones are important to mast cell density in the CNS as illustrated by the fact that even in naïve control females, mast cells appear to be regulated by gonadal hormones. Fewer mast cells are located in select thalamic nuclei of ovariectomized (OVX) mice than in intact, sham-operated females (Kovács and Larson, 2002) and estrogen restores these populations in OVX females (Kovács and Larson, 2003). Reproductive hormones influence mast cells via hormonal receptors (Wilhelm et al., 2000). While estrogen alone does not influence mast cell activity, it increases the tendency for mast cells to degranulate in response to compounds such as substance P (Rozniecki et al., 1999; Vliagoftis et al., 1992) while testosterone exerts a stabilizing influence (Vliagoftis et al., 1992).
Several thalamic nuclei, including the VPM, the VPL, and Po receive nociceptive input (Al-Chaer et al., 1996; Diamond et al., 1992). In primates and rodents these nuclei receive projections of spinothalamic neurons from the dorsal horn (Battaglia et al., 1992; Graziano and Jones, 2004; Li et al., 1996; Willis and Westlund, 1997) and transmit nociceptive signals primarily to areas including the somatosensory cortex. While the Po receives input from the whole body, the VPM is somatotopically organized with input primarily from the head whereas the VPL is the target for tactile and nociceptive input from the trunk and limbs. Spinothalamic neurons express substance P (Battaglia and Rustioni, 1992; Battaglia et al., 1992; Nishiyama et al., 1995) and are positive for neurokinin-1 receptor immunoreactivity in rats (Li et al., 1996; Marshall et al., 1996) and monkeys (Yu et al., 1999). Substance P is not only a potent degranulator of mast cells in the dura (Ottosson and Edvinsson, 1997) and periphery (Foreman, 1987; Marshall et al., 1994; Singh et al., 1999), it is also chemotactic to mast cells (Ottosson and Edvinsson, 1997; Rozniecki et al., 1999). Other pro-nociceptive compounds, such as NGF, have similar chemotactic properties (Sawada et al., 2000). In addition, NGF, dynorphin, and TNF-α, whose neuronal expressions are upregulated following nerve injury, are capable of triggering the degranulation of mast cells (Horigome et al., 1993; Sugiyama and Furuta, 1984; Sydbom and Terenius, 1985).
The number of visible mast cells depends on a balance between recruitment of mast cells (chemotaxis) and the loss of granule contents (degranulation). It is not clear how these two dynamic processes result in the relative increase in thalamic mast cell populations contralateral to the SNL. When examined at the level of thalamic nuclei, the asymmetry was statistically significant in the Po and LG nuclei. However, these differences were small, contributing little to the large difference between thalamic hemispheres. While the cue recruiting mast cells to the contralateral LG is not known, substance P released from spinothalamic neurons in response to nociception (Guilbaud et al., 1977) may be responsible for the increased number of mast cells in the Po. However, the density of mast cells in the VPL after both SNL and sham-surgery is low in spite of intense nociceptive input. One might speculate that this reflects the tendency for both SNL as well as sham surgical procedures to activate spinothalamic tract neurons. The mechanical manipulation or inflammation following surgery may induce release of substance P and depletion of mast cells by a massive degranulation. The differential distribution of mast cells amongst nuclei with spinothalamic input is not understood but may be relevant to the unique role of each nucleus in the processing of SNL nociceptive activity.
When mast cell density is evaluated without respect to hemispheric distribution, the only nucleus in which the relative mast cell population differed in both hemispheres of SNL mice compared to sham-operated controls was the PF of females. There, the density of mast cells after SNL was less than in historical control mice. The PF participates in somatosensory, motor and nociceptive activity, receiving projections from the somatosensory and motor cortices (Cornwall and Phillipson, 1988). Stimulation of the PF produces pain-like behavior in animals (Rosenfeld and Holzman, 1978). Thus, differences in the relative population of mast cells in the PF between SNL and sham-operated females are probably associated with the hyperalgesic effect of SNL and are deserving of further study.
5. Summary
These data indicate that thalamic mast cells, in female but not male mice, are responsive to nerve injury-evoked nociceptive signals, suggesting a potential involvement of thalamic mast cells in the integration of nociceptive processing. SNL induced an asymmetric distribution of mast cells in females that is consistent with the spinothalamic projection of nociceptive signals from the site of injury to the contralateral thalamus. The asymmetry of mast cells in females after SNL suggest that nociceptive input rather than stress resulting from noxious stimulation is responsible for these effects.
Acknowledgements
This work was supported by the National Institutes of Health grant NS39740 (A.A.L.) funded by the National Institute of Neurological Disorders and Stroke and the National Institutes on Arthritis and Musculoskeletal and Skin Diseases. The authors wish to thank Christopher Hall for his excellent technical assistance and Dr Carolyn Fairbanks for her helpful review of this work.
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Keywords:
Thalamus; Mast cells; Estrogen; Allodynia; Hyperalgesia; Dimorphic
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