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1Departments of Surgery and 2Neuroscience, Brown Medical School, Rhode Island Hospital, Providence, Rhode Island
Submitted 13 June 2005; accepted in final form 24 July 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Wakefield and Chang 2005). Although the exact trigger is not certain, proinflammatory cytokines are elevated in the aqueous and vitreous humors in recurrent uveitis (Di Girolamo et al. 1996; El-Shabrawi et al. 2000). Acute anterior uveitis can be produced experimentally in animal models by a single injection of endotoxin or lipopolysaccharide (LPS) at a peripheral site remote from the eye (Rosenbaum et al. 1980; see Smith et al. 1998). Because endotoxin-induced uveitis (EIU) mimics many of the early events in anterior uveitis, including a breakdown of the blood-aqueous barrier and leukocyte accumulation, it has been used extensively to examine the peripheral cellular and biochemical mechanisms associated with ocular inflammation (Bhattacherjee et al. 1983; Cousins et al. 1984; Cuello et al. 2002; Herbort et al. 1988; Planck et al. 1994). However, the central neural correlates of ocular inflammation in uveitis have received little attention.
It is well established that persistent or recurrent tissue inflammation alters the properties of spinal dorsal horn neurons that contribute to behavioral hyperalgesia (see Kidd and Urban 2001; Treede et al. 1992; Woolf and Salter 2000). Although acute injury to craniofacial tissues sensitizes trigeminal brain stem neurons (see Sessle 2000), no study has assessed the effects of intraocular inflammation on second-order neurons that receive sensory input from the anterior eye. The ophthalmic branch of the trigeminal nerve supplies nearly all tissues of the eye including the ocular surface, iris muscle, and ciliary body (Beckers et al. 1992; Belmonte et al. 1997; Marfurt et al. 1989; ten Tusscher et al. 1989). Trigeminal fibers that innervate the eye terminate centrally in the lower portions of trigeminal brain stem complex, mainly in trigeminal subnucleus caudalis (Vc) (Gong et al. 2003; Marfurt 1981; Marfurt and del Toro 1987; Panneton and Burton 1981). Animal models of trigeminal pain often have focused on the role of Vc because this laminated subnucleus shares several properties with the spinal dorsal horn (Bereiter et al. 2000; Dubner and Bennett 1983; Sessle 2000). Evidence obtained in rats indicate that the ocular surface (e.g., cornea/conjunctiva) is represented at two spatially distinct regions of Vc, a rostral trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition region and a caudal subnucleus caudalis/upper cervical spinal cord (Vc/C1) junction region (Gong et al. 2003; Lu et al. 1993; Marfurt and del Toro 1987; Meng and Bereiter 1996; Strassman and Vos 1993). Considerable evidence from naïve rats indicates that cornea/conjunctiva units at the Vi/Vc transition and Vc/C1 junction regions have different encoding properties, responses to opioid analgesics, and efferent projections (Hirata et al. 1999, 2000; Meng et al. 1997, 1998). This has led to the hypothesis that different portions of Vc serve different aspects of trigeminal function in acute ocular pain (Bereiter et al. 2000; Hirata et al. 2004). The aim of the present study was to determine the effects of persistent ocular inflammation, as produced in the EIU model, on sensory encoding by second-order cornea/conjunctiva units at the Vi/Vc transition and Vc/C1 junction regions. In parallel experiments, evoked tear volume and eye blink frequency were measured as indices of ocular-specific reflex function during EIU-induced inflammation. The general hypothesis tested in this study is that intraocular inflammation differentially modulates the properties of neurons in different portions of Vc and, in turn, the magnitude of selected ocular-specific reflexes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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EIU model
Male rats (250–410 g, initial body wt, Sprague-Dawley, Harlan) were given a single injection of endotoxin (LPS, 1 mg/kg ip, Salmonella typhimurium, Sigma, St. Louis, MO), dissolved in sterile saline. Animals survived either 2 or 7 days after LPS; body weight was monitored daily. Any ocular or periocular pathology was noted on the day of the experiment (i.e., crusting, hyperemia), and under deep barbiturate anesthesia a sample of aqueous humor was collected from the anterior chamber with a 30-gauge needle under microscopic guidance after the recording session. The total aqueous humor sample (4–7 µl) was split, and 1–2 µl was fixed on a glass slide and stained with hematoxylin-eosin to assess accumulation of leukocytes. Cell counts were scored without prior knowledge of treatment: 0 = 0–3 cells, 1 = 3–10 cells, and 2 = >10 cells/sample. The remainder of the aqueous humor sample was used to measure total protein by the Bradford assay (Pierce BCA Kit, Rockford, IL).
Surgical preparation for electrophysiology
Animals were anesthetized with pentobarbital sodium (70 mg/kg ip) during all surgical procedures. The right femoral artery and left jugular vein were catheterized for monitoring blood pressure and infusing drugs, respectively. The animals were tracheotomized and respired artificially with oxygen-enriched room air. The rat was placed in a stereotaxic frame and portions of the occipital bone and C1 vertebra were removed to expose the dorsal surface of the caudal brain stem and upper cervical spinal cord. The brain stem surface was covered with warm mineral oil. Anesthesia was maintained after surgery by a continuous infusion of pentothal sodium (20–25 mg · kg–1 · h–1) and switched to a mixture of pentothal sodium and the short-acting paralytic agent, gallamine triethiodide (25 mg · kg–1 · h–1), only immediately prior to neural recording. Expiratory end-tidal CO2 was monitored continuously and kept at 4–5% by adjusting tidal volume. Mean arterial pressure (MAP) remained >100 mmHg throughout all experiments. Body temperature was maintained at 38°C with a heating blanket and a rectal thermal probe.
Electrophysiology recording techniques
Single neurons were recorded extracellularly at the Vi/Vc transition and laminae I–II at the Vc/C1 junction regions with tungsten electrodes (9–15 M
, FHC, Bowdoinham, ME) as described previously (Hirata et al. 1999, 2003; Meng et al. 1997). A few units (<10) were found, after histological confirmation, to have been recorded from deep laminae (laminae III–V) at the Vc/C1 junction; however, the results from these experiments were not included in subsequent analyses. Unit activity was amplified, displayed on a digital oscilloscope to monitor spike shape and amplitude, and passed through a window discriminator. Discriminated neural spikes, MAP and a marker for stimulus onset were acquired and displayed on-line with an Apple computer (G3) through a DAQ interface board using LabVIEW software (National Instruments, Austin, TX). Data also were recorded on a four-channel DAT/SCSI-based acquisition system (Cygnus Technology) for off-line analyses.
Characterization of corneal units
A fine camel hair brush was applied gently to the ocular surface (e.g., corneal surface and conjunctiva) as a search stimulus. Units responsive to mechanical stimulation of the ocular surface, referred to as "ocular units," were tested for convergent cutaneous input with calibrated von Frey filaments, press and then light pinch of facial skin. Ocular units with an excitatory cutaneous receptive field (RF) were further classified as low-threshold mechanoreceptive (LTM), wide dynamic range (WDR), or nociceptive specific (NS) as described previously (Hu 1990; Meng et al. 1997). The high-threshold excitatory RF area of WDR and NS units was mapped by light pinch of facial skin using a small forceps onto a standardized series of drawings of the rat face. Some units found only at the Vi/Vc transition were classified as "complex units," characterized by a large convergent inhibitory cutaneous RF that was contiguous with the cornea/conjunctiva border (see Hirata et al. 2004). Other ocular units found only at the Vi/Vc transition had no apparent cutaneous RF and were classified as cornea only (CO). Because all units were activated by mechanical stimulation of the palpebral conjunctiva of the lower eyelid, the threshold for this input was determined with von Frey filaments. Most units were excited by mechanical stimulation of the corneal surface and conjunctiva; however, some units at the Vi/Vc transition (7/74) and Vc/C1 junction (23/107) regions were excited by conjunctiva stimulation alone. Post hoc analyses revealed no significant differences in mechanical threshold, cutaneous RF area, or responses to topical application of chemical stimulants to the ocular surface between units with cornea and conjunctiva input versus conjunctiva input alone. Thus the responses of these cells with conjunctiva only and conjunctiva plus cornea RFs were grouped for further statistical analyses.
Ocular surface stimulation by chemical agents
Units were tested with one of three chemical stimulants: histamine, nicotine, or CO2 gas applied to the ocular surface in separate experimental preparations. Cells responsive to at least one of these chemical agents were included in further data analyses. The majority of units were tested with histamine (1 or 10% histamine-HCl, 10 µl, pH 7.4, dissolved in rat artificial tear fluid) (see Kessler et al. 1995) applied by micropipette to the dorsal cornea-conjunctiva border. This volume of histamine (10 µl) was sufficient to cover the ocular surface yet remained in place by surface tension without spilling over to the periorbital skin below the eye. At least 15 min elapsed between each application of a chemical test agent. Histamine was used as a test agent since it is a key contributor to allergic conjunctivitis and other ocular irritation conditions (Minami and Kamei 2004). Units were tested in a similar manner with nicotine (1 or 10%, dissolved in 0.9% saline, pH 7.1, 10 µl). Nicotine produces sharp pain in humans when applied to the nasal mucosa (Thuerauf et al. 1999) and may excite selective subpopulations of ocular afferent nerves in animals (Tanelian 1991).
The noxious chemical stimulant, CO2 gas, was applied to the ocular surface by a computer-controlled system adapted from the method of Chen et al. (1995). Concentrations of CO2 gas >35% reliably produce a sharp stinging pain when applied to the ocular surface in humans (Acosta et al. 2001). Variable concentrations (0, 30, 60, and 80%) of CO2 gas were obtained by mixing the outflow from tanks of 100% CO2 and air through a proportional gas mixer as monitored from the bleeder valve output by an infrared detector (CapStar 100, CWE, Ardmore, PA). Humidified gas mixtures were delivered at a constant flow rate (200–500 ml/min) to the left eye. The timing and duration of CO2 pulses (40-s duration, minimum of 15 min between pulses) were controlled by LabVIEW software as described previously (Hirata et al. 1999). The ocular surface was kept moist with a pH balanced artificial rat tear fluid during surgery and between stimulus periods throughout the experiment.
In several animals, the properties of units at the Vi/Vc transition (n = 4) or Vc/C1 junction region (n = 4) were tested during development of acute inflammation of the ocular surface induced by the selective small fiber excitant, mustard oil (MO, 20% solution), or the inflammatory agent croton oil (3% solution) applied topically to the ocular surface. In a few cases (n = 3), the acute effects of LPS (1 mg/kg ip) were assessed before and 4 h after injection.
CO2 tear volume
Tear volume was measured in response to a graded series of CO2 pulses (40-s duration) in a separate group of rats, prepared as for neural recording, placed in a stereotaxic frame and maintained under pentothal sodium anesthesia (20–25 mg · kg–1 · h–1 iv). Tear volume was determined by the change in weight of a filter paper strip (
5 x 8 mm) in contact with the cornea/conjunctiva for 2 min/sample. The filter paper was positioned at the inferior-lateral edge of the cornea-conjunctiva interface that allowed tear volume to be determined while applying CO2 pulses to the center of the cornea. Each paper strip was weighed and then placed at the cornea-conjunctiva border 1 min prior to and removed 1 min after the onset of the CO2 pulse. The ocular surface was kept moist with artificial tears during the interstimulus intervals (15 min) and excess fluid was removed just prior to placement of the filter paper for subsequent samples (Hirata et al. 2004).
CO2 eye blink
Male rats were anesthetized with urethan (1.2 g/kg ip) and allowed to breathe spontaneously. Urethan was used because cornea stimulation-evoked blinks under barbiturate anesthesia often produces incomplete or "flutter" like responses that are difficult to quantify by videotape analyses (personal communication, Dr. Craig Evinger). Fifteen minutes after anesthesia, the rat was placed in a stereotaxic frame using gentle insertion of blunt ear bars and incisor bar for 
30 min prior to stimulation. Adequate depth of anesthesia was confirmed in each animal by absence of a withdrawal reflex to strong pinch of the hindlimb. Eye blinks were evoked by a graded series of CO2 pulses (40-s duration) delivered every 15 min to the ocular surface as described in the preceding text for unit recording. Blinks were captured from the video output of a surgical microscope (Zeiss, Thornwood, NY) and A/D converter (Dazzle, Fremont, CA) onto a Macintosh computer (G4) using iMovie software (Apple), and the recorded display was analyzed off-line at slow speed. A rapid lid movement resulting in closure of more than 20% was counted as a blink and latency was defined as the time to first blink after stimulus onset.
Data analysis
Neural recording data were acquired and displayed by LabVIEW as peristimulus time histograms (PSTH) of spikes per 1-s bins, exported to a spreadsheet and analyzed off-line. The responses to histamine, nicotine, and CO2 gas were determined from the average spontaneous activity rate (SA, spikes/s) collected 30 s immediately prior to each test stimulus. As described previously (Hirata et al. 1999, 2003) the total response magnitude (Rmag) for each stimulus period was defined as the cumulative sum of spikes for contiguous bins in which the spike count exceeded the mean +2 SD of the background activity. A total Rmag was calculated for each stimulus period and can be thought of as equivalent to the "area under the curve." Total Rmag values across a range of stimulus concentrations of histamine, nicotine, and CO2 gas for units recorded in naïve and 2- and 7-day LPS-treated rats were assessed by ANOVA corrected for repeated measures (Winer 1971) and individual comparisons by Newman-Keuls. The high-threshold cutaneous RF areas of WDR and NS units were mapped onto standardized drawings of the rat face, digitized and quantified by a planimetric method using National Institutes of Health Image software (v. 1.61). 
2 analysis determined if the overall proportion of WDR and NS units among histamine-, CO2-, and nicotine-positive neurons at the Vc/C1 junction was similar for naïve and 2- and 7-day LPS-treated rats. Other properties of corneal units (i.e., spontaneous activity, cutaneous RF area, von Frey threshold) as well as tear volume and eye blink after CO2 stimulation were assessed by ANOVA and corrected for repeated measures when appropriate. 2 analysis determined the likelihood of observing leukocyte accumulation in the anterior eye after LPS.
Histology
At the end of the recording session, the rat was given a bolus of pentobarbital sodium (60 mg/kg iv) and perfused through the heart with saline followed by 10% formalin. Brain stem sections were cut at 40 µm on a freezing microtome and stained with cresyl violet. Recording sites at the Vi/Vc transition and Vc/C1 junction regions were marked electrolytically (5 µA, 10 s).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Body weight was reduced significantly (4.5 ± 0.7%, P < 0.001) 2 days after LPS (initial = 322 ± 5 g, 2-d post-LPS = 306 ± 4 g, n = 52), whereas by 7 days, body weight was similar to the initial value (initial = 328 ± 5 g, 7-d post-LPS = 332 ± 4 g, n = 48). Body weight averaged 342 ± 5 g (n = 69) for naïve rats. LPS had a noticeable effect on the general appearance of the eye. At 2 days after LPS, most rats had hyperemia of the sclera and a reddish exudate at the eyelid/conjunctiva edge, whereas by 7 days after LPS, the eye appeared normal. At the end of some experiments, an aqueous humor sample was collected and scored for the presence of leukocytes and/or measured for total protein. The aqueous humor from all 2-day LPS-treated rats examined (n = 23) contained a moderate to high number of leukocytes; however, in 7-day LPS-treated rats, cells were seen in only 2 of 17 animals and no cells were seen in samples from naïve rats (n = 20), a highly significant difference by 2 analysis (P < 0.001, 2 = 28.7, df = 4). The protein content of aqueous humor of 2-day LPS-treated rats was elevated significantly (0.93 ± 0.09 mg/ml, P < 0.05, n = 6) compared with naive (0.55 ± 0.08 mg/ml, n = 6) or 7-day LPS-treated animals (0.23 ± 0.1 mg/ml, n = 4).
Recording sites
All recovered lesions were plotted as a function of LPS treatment for recording sites at the Vi/Vc transition (top) and Vc/C1 junction regions (bottom) regardless of the chemical agent tested (i.e., histamine-, nicotine-, and CO2-responsive units) as shown in Fig. 1. Recording sites at the Vc/C1 junction were restricted to the superficial laminae within 200 µm of the dorsal brain stem surface. Sites recovered at the Vi/Vc transition were clustered at the ventral pole of the nucleus. No obvious difference in distribution of recording sites for the three treatment groups was seen.
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HISTAMINE-INDUCED UNIT ACTIVITY.
Ninety-one neurons in laminae I–II at the Vc/C1 junction region were tested for responsiveness to histamine applied to the ocular surface. The proportion of Vc/C1 units activated by histamine was affected by LPS treatment (P < 0.05, 2 = 7.71, df = 2). In naïve rats 24 of 26 units were excited by histamine (1 or 10% solution). However, at 2 days after LPS, only 28 of 40 units responded to histamine, whereas 7 days after LPS, 23 of 25 units were responsive. Low dose (1%) histamine was sufficient to excite 22 of 24 units at the Vc/C1 junction in naïve rats, but only 10 of 28 units in 2-day LPS-treated animals, whereas all 23 units were activated in the 7-day LPS-treated group. As shown in Fig. 2, histamine-evoked activity was characterized by a rapid increase in firing rate after 1% histamine and a sustained increase in firing rate after 10% histamine in naïve rats (top). The sustained increase in evoked activity was most pronounced in the 7-day LPS-treated group after 1% and 10% histamine (Fig. 2, bottom), whereas both the peak firing rate and response duration were greatly reduced in 2-day LPS-treated animals compared with the naive group (Fig. 2, middle). The average total Rmag (spikes per stimulus above background, see METHODS) to histamine for all Vc/C1 units is shown in Fig. 3 (top). Analyses of these data by cell class indicated that LPS had a similar effect on histamine-evoked total Rmag of WDR (Fig. 3, middle) and NS units (Fig. 3, bottom). However, the increase in total Rmag for WDR units of 7-day LPS-treated animals to 10% histamine was significantly greater than NS units (P < 0.01). Response duration to 1 and 10% histamine of units from 2-day LPS-treated rats averaged 12.5 ± 2.4 and 23.9 ± 4.7 s and was significantly reduced (P < 0.01) compared with the naïve group (1% = 21.4 ± 3.4 s and 10% = 35.2 ± 3.5 s). Histamine-evoked response duration in 7-day LPS-treated rats was increased significantly (P < 0.01) to 38.9 ± 3.2 and 47.6 ± 3.6 s, respectively, after 1 and 10% histamine versus the naïve group. LPS-induced changes in response duration to histamine were similar for WDR and NS units.
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Chemical stimulation of ocular units at the Vi/Vc transition region
HISTAMINE-INDUCED UNIT ACTIVITY. Nearly all units tested at the Vi/Vc transition were activated by histamine regardless of LPS treatment (naïve = 15/16; 2-day LPS = 25/27; and 7-day LPS = 24/28 units). The lower concentration of histamine (1%) was sufficient to activate 14 of 15 units at the Vi/Vc transition from naïve subjects, 23 of 25 units after 2-day LPS, and 23 of 24 units after 7-day LPS treatment. The average histamine-evoked total Rmag for units at the Vi/Vc transition, independent of cell class, for each group is shown in Fig. 5 A. Total Rmag was similar for units from naïve and 7-day LPS-treated rats; however, units from 2-day LPS-treated rats were reduced significantly (P < 0.01). Although many units at the Vi/Vc transition had unique RF properties not generally seen for units at the caudal Vc/C1 junction (i.e., CO and complex units), further analysis of histamine-evoked responses for Vi/Vc units by cell type revealed only minor group differences in histamine-evoked total Rmag of naïve and 2- and 7-day LPS-treated rats (data not shown). Histamine-evoked (10% solution) response duration of units from naïve rats (42.6 ± 7 s) was not different from units in 2-day (35 ± 4.9 s) or 7-day LPS-treated rats (44.2 ± 6.4 s).
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EFFECT OF ACUTE INFLAMMATION ON OCULAR SURFACE-EVOKED UNIT ACTIVITY. The effect of acute inflammation of the ocular surface on subsequent evoked responses was examined 4 h after mustard oil or croton oil was applied topically to the ocular surface while recording from histamine-responsive units at the Vc/C1 junction (n = 4) or Vi/Vc transition (n = 4). The total Rmag to 10% histamine for Vc/C1 units averaged 277 ± 104 and 233 ± 150 (SE) spikes/stimulus before and 4 h after inflammation, respectively. Similarly, the total Rmag to 10% histamine for Vi/Vc units averaged 510 ± 300 and 260 ± 187 spikes/stimulus before and 4h after inflammation, respectively. Although the initial firing rate of Vc/C1 (4.9 ± 1.2 spikes/s) and Vi/Vc units (1.1 ± 0.6 spikes/s) increased significantly (P < 0.01) immediately after acute inflammation, by 4 h, it was not different from the initial rate. In three additional rats, laminae I–II units at the Vc/C1 junction were tested before and 4 h after LPS (1 mg/kg ip) and in each case the magnitude of nicotine-evoked activity was similar to the control response (data not shown).
SPONTANEOUS ACTIVITY AND MECHANICAL RF PROPERTIES OF CORNEAL UNITS.
LPS treatment did not alter the overall proportion of laminae I–II units at the Vc/C1 junction classified as WDR or NS (P > 0.05, 2 analysis). The average spontaneous activity of WDR and NS units at the Vc/C1 junction for all histamine-, CO2-, and nicotine-responsive units revealed a small but significant (P < 0.05) increase 7 days after LPS among WDR units (10.5 ± 1.3 spikes/s, n = 19) compared with units from naïve (4.1 ± 0.5 spikes/s, n = 20) and 2-day LPS-treated rats (7.6 ± 1.8 spikes/s, n = 19). The spontaneous activity of NS units in laminae I–II at the Vc/C1 junction was not affected significantly by LPS treatment (naïve = 4.7 ± 1.1, n = 16; 2-day LPS = 4.3 ± 1.3, n = 18; 7-day LPS = 5.9 ± 1.6 spikes/s, n = 16). The spontaneous activity of units at the Vi/Vc transition region classified as complex units increased (P < 0.05) 2 days after LPS (24.4 ± 11.3 spikes/s, n = 3) compared with complex units in naïve rats (9.5 ± 2.9 spikes/s, n = 7) or in 7-day LPS-treated rats (12. 7 ± 5.2 spikes/s, n = 7). The spontaneous activity of Vi/Vc units classified as "cornea only" was reduced (P < 0.025) 2 days after LPS (1.1 ± 0.8 spikes/s, n = 12) compared with the naïve group (6.7 ± 3.7 spikes/s, n = 8) or 7-day LPS-treated rats (3.6 ± 1.3 spikes/s, n = 7). The spontaneous activity of Vi/Vc units classified as WDR (9.4 ± 6.6 spikes/s, n = 6) or NS (8.6 ± 7.2 spikes/s, n = 4) in naïve rats was variable and not affected by LPS treatment.
The high-threshold cutaneous RF area of caudal Vc/C1 ocular units was mapped and analyzed separately for units classified as WDR and NS. The cutaneous RF of each Vc/C1 unit was contiguous with the ocular surface. The average RF area of WDR units in 7-day LPS-treated animals was greater than naïve or 2-day LPS-treated rats (P < 0.01, Fig. 6). The RF area of NS units was smaller than WDR units in naïve (P < 0. 05) and 7-day LPS-treated rats (P < 0.01) and did not change after LPS (P > 0.05). The cutaneous RF area of ocular units at the Vi/Vc transition classified as WDR or NS generally were larger than those of Vc/C1 units and did not change consistently after LPS treatment (range = 1.2 –9.3 cm2).
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CO2-INDUCED TEAR VOLUME. Spontaneous tear volume was low (0.3 ± 0.1 mg/2 min, n = 12–23 per group) in all treatment groups. Stimulation of the ocular surface by room air (0% CO2) caused a small but significant increase (1.2 ± 0.2 mg/2 min, P < 0.01) compared with the spontaneous volume. As shown in Fig. 7, tear volume increased with higher concentrations of CO2 in naive and 7-day LPS-treated rats; however, 2-day LPS-treated animals displayed little or no increase evoked tear volume above that seen after stimulation with 0% CO2. This was a consistent finding as only 1 of 12 2-day LPS-treated rats had an evoked tear volume to 80% CO2 that exceeded the response to 0% CO2 by >50%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Cousins et al. 1984; Rosenbaum et al. 1980). The pronociceptive cytokines, TNF
, and IL-6, increase transiently in aqueous humor over the first 4 h and again at 24 h after LPS and return to control levels by 48h (de Vos et al. 1994). Although some agents such as IL-1
(Planck et al. 1994), prostaglandin E2, and substance P may remain elevated in the aqueous for 
72 h (Herbort et al. 1988), there is little evidence of intraocular inflammation or elevated cytokine levels in the aqueous 7 days after a single nonlethal dose of LPS in the rat model for EIU. The high dose of LPS used here (1 mg/kg), necessary to reliably cause EIU (Cousins et al. 1984), also produces a wide variety of systemic effects (e.g., fever, reduced locomotor activity, elevated plasma levels of cytokines); however, these effects generally resolve after 1–2 days (Azab and Kaplanski 2004; Givalois et al. 1994; Romanovsky et al. 1996). Pain-like behavior in awake animals also is modified transiently by similar doses of LPS and return to normal within 1–3 days. For example, visceral sensitivity is increased after 1 mg/kg LPS, lasts only 9–12 h and returns to normal after 1 day (Coelho et al. 2000; Ishihawa et al. 1989). Cutaneous sensitivity as seen by hotplate latency increases rapidly (1–2 h) after LPS, develops into hypoalgesia by 4 h, and returns to normal by 20 h (Yirmiya et al. 1994). Grip force is greatly reduced after 0.9 mg/kg LPS in mice for 3–6 h, in a morphine-reversible manner, but is normal by 24 h (Kehl et al. 2004). Collectively, these results suggest that the effects of systemic LPS on pain behavior evoked from elsewhere in the body is less pronounced than the effects on the ocular units and ocular-related reflexes seen in the present study. Indeed, in their original report of the EIU model, Rosenbaum et al. (1980) described persistent signs of inflammation in the eye after LPS that were more severe and lasted longer than responses in other tissues and were not due to preferential uptake of LPS by ocular tissues (Rosenbaum et al. 1983). The basis for the pronounced effects of LPS on the eye is not known; however, it is possible that the pattern and/or actions of cytokines may have contributed to the persistent effects on ocular-related neural pathways. For example, unlike other tissues in which a cascade-like pattern of cytokine production is seen, pro-inflammatory cytokines increase simultaneously in the eye (de Vos et al. 1996). A simultaneous increase in multiple cytokines may have acted synergistically to exaggerate an initial barrage of primary afferent activity that, in turn, lead to the persistent central sensitization seen 7 day after LPS. The action of specific cytokines in the eye also may be different from in other tissues. Systemic anti-TNF antibody treatment prevents LPS-induced inflammation in other tissues but has little effect on the signs of uveitis, and further, if injected directly into the eye, even exacerbates the signs of ocular inflammation (de Vos et al. 1995). The EIU model also produces effects on ocular inflammation and reflexes that differ from models of direct injury to the ocular surface. Silver nitrate cauterization of the cornea causes leukocyte accumulation in corneal stroma over 6 h and elevated cell counts are seen at 48 h; however, capsaicin-evoked blinks are increased only for 12 h compared with naïve rats (Wenk and Honda 2003).
Several lines of evidence suggest that central neural mechanisms play a dominant role in modulation of unit activity and ocular-related reflexes during EIU. First, ocular units at the Vi/Vc transition and Vc/C1 junction regions displayed differential responses to ocular stimulation. At 2 days after LPS, ocular unit activity was reduced at both regions: however, at 7 days after LPS, only units at the Vc/C1 junction displayed enhanced responses despite the fact that ocular units at both regions receive a common input and have similar thresholds to chemical or mechanical stimulation (Hirata et al. 1999; Meng et al. 1997). Second, neither mechanical nor chemical thresholds for ocular units at either region were reduced significantly by LPS treatment. Also, the latency for CO2-evoked eye blink in 2- or 7-dday LPS-treated rats was not different from naïve animals. A reduction in threshold for second-order neurons or reduced latency for evoked reflexes would suggest modulation of primary afferent sensitivity (see Kidd and Urban 2001; Treede et al. 1992; Woolf and Salter 2000). Because maintenance of peripheral sensitization of primary afferent neurons requires continuous exposure to proinflammatory agents (Boylard et al. 2000), and ocular inflammation is minimal at 7 days after LPS, it seems unlikely that the enhanced responsiveness of Vc/C1 ocular units at 7 days is due mainly to peripheral mechanisms and ongoing intraocular inflammation. Third, convergent cutaneous RF areas of WDR units at the Vc/C1 junction, but not at the Vi/Vc transition region, were enlarged 7 days after LPS. It is well established that enlargement of cutaneous RF area normally occurs through central mechanisms (Cook et al. 1987; Dubner 1992; Jinks and Carstens 1998). However, it cannot be excluded that the RF area of primary afferent fibers also were enlarged after LPS (see Bereiter and Barker 1980). Although the pharmacological basis for long-term modulation of corneal units during EIU was not addressed here, preliminary studies suggested that increased GABAA receptor activity may have contributed to the reduction in evoked corneal unit activity and reflex lacrimation 2 days after LPS because topical application of the selective GABAA receptor antagonist, bicuculline methiodide, partially reversed these responses (Bereiter et al. 2004). Increased GABAA activity has been associated with presynaptic mechanisms that produce allodynia during persistent inflammation (Weng et al. 1998).
Ocular unit sensitivity was tested against different chemical agents applied to the ocular surface. This was necessary because selectivity for different classes of algesic agents has been reported for corneal afferent nerve fibers (Chen et al. 1997; Tanelian 1991). Although H1 receptor-positive neurons are found in significant numbers in the trigeminal ganglion (Kashiba et al. 1999) and contribute to intraocular inflammation in EIU (Yamahiro et al. 2001), histamine normally evokes itch rather than pain when injected into the skin (Schmelz et al. 2003). Thus it was important to test ocular units with agents known to cause pain in humans such as CO2 gas (Chen et al. 1995) and nicotine (Thuerauf et al. 1999). Individual units were tested with only one chemical here; however, in a similar study in naïve rats, Carstens et al. (1998) compared the responses of 18 units in superficial laminae at caudal Vc to ocular application of low pH buffer, nicotine, and histamine and found that 16/18 units responded to at least two of the three agents. Simone et al. (2004) reported that all spinothalamic tract cells in the primate dorsal horn that were activated by histamine also were excited by capsaicin (however, see Andrew and Craig 2001). These results suggested that modulation of ocular units during EIU was not selective for unique classes of chemosensitive ocular afferent nerves.
The finding that ocular units at the Vi/Vc transition and Vc/C1 junction regions displayed different response patterns during EIU was consistent with the hypothesis that these regions mediate different aspects of ocular function (see Bereiter et al. 2000). Previously, we reported that ocular units at the Vi/Vc transition, but not at the Vc/C1 junction, were necessary for control of reflex lacrimation in naïve rats (Hirata et al. 2004). The present results supported that conclusion because the response pattern for both Vi/Vc units and reflex lacrimation were reduced at 2 days and returned to normal by 7 days after LPS. By contrast, the response pattern of ocular units at the Vc/C1 junction and evoked eye blink frequency were similar in that both were enhanced 7 days after LPS. The caudal portion of Vc is necessary for the R2 component of the blink reflex, a component mediated by small A
afferent fibers (Pellegrini et al. 1995). It is well accepted that the caudal Vc is critically involved in neuroplasticity and central sensitization after craniofacial tissue injury (Chiang et al. 2002; Sessle 2000). Because neurons in the lateral portions of the caudal Vc/C1 junction region often receive convergent input from the ocular surface, nasal mucosa, and meninges (Burstein et al. 1998; Peppel and Anton 1993; Schepelmann et al. 1999), this region may contribute to a wide variety of clinical conditions (e.g., dry eye, uveitis, sinusitis, migraine) that result in referred pain.
These results suggest that the neural pathways for ocular sensory processing are overly sensitive to systemic infection as seen by the long-term changes in medullary dorsal horn neurons during EIU. Understanding the role of central neurons in ocular inflammatory disorders such as uveitis and dry eye syndrome (van Bijsterveld et al. 2003) may shed new light on management of ocular conditions that have traditionally been viewed from a peripheral perspective.
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Address for reprint requests and other correspondence: D. A. Bereiter, Brown Medical School, Rhode Island Hospital, Depts. of Surgery and Neuroscience, 222 Nursing Arts Bldg., Providence, RI 02903-4970 (E-mail: David_Bereiter{at}brown.edu)
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), 2-day LPS-treated (
), and 7-day LPS-treated (
) rats. Top: all units in naïve (n = 24), 2-day LPS (n = 28), and 7-day LPS treatment groups (n = 23); middle: WDR units (naïve = 14, 2-day LPS = 12, 7-day LPS = 11); bottom, NS units (naïve = 10, 2-day LPS = 16, 7-day LPS = 12). **P < 0.01 vs. 0% histamine; a = P < 0.05, b = P < 0.01 vs. naïve group. Test periods separated by 15 min.
