[1] 11w Skin denervation, neuropathology, and neuropathic pain in a laser-induced focal neuropathy

[1] 418w Small-diameter sensory nerves innervating the skin are responsive to noxious stimuli, and an injury to these nerves is presumably related to neuropathic pain. Injury-induced neuropathic pain in animals can be produced by laser irradiation, which usually requires concomitant use of photosensitive dyes, known as the photochemical approach. It is not clear whether laser irradiation alone can induce neuropathic pain. In addition, two issues are important to apply these approaches: the relationship between the extent of laser irradiation and the occurrence of neuropathic pain, and the susceptibility of small-diameter sensory nerves in the skin to laser-induced neuropathic pain. To address these issues, we designed a new model of focal neuropathy by applying a diode laser of 532 nm (100 mW) to the sciatic nerve and evaluated small-diameter nerves by quantifying skin innervation and largediameter nerves by measuring amplitudes of the compound muscle action potential (CMAP). Immediately after laser irradiation, epineurial vessels were occluded due to the formation of thrombi, and the blood flow through these vessels was markedly reduced. On postoperative day (POD) 2, animals developed characteristic manifestations of neuropathic pain, including spontaneous pain behaviors, thermal hyperalgesia, and mechanical allodynia. These phenomena peaked during PODs 7-21, and lasted for 3-6 weeks. The neuropathology at the irradiated site of the sciatic nerve included a focal area of axonal degeneration surrounded by demyelination and endoneurial edema. The extent of damage to large-diameter motor and sensory nerves after laser irradiation was evaluated by nerve conduction studies. On the irradiated sides, amplitudes of the compound muscle action potentials and sensory nerve action potentials (SNAPs) were reduced to 65.0% (P b 0.0001) and 42.5% (P b 0.01) of those on the control sides, respectively. Motor innervation of the neuromuscular junctions (NMJs) on plantar muscles was examined by combined cholinesterase histochemistry and immunohistochemistry. The ratio of innervated NMJs on the operated sides decreased to 76.3% of that on the control side. Skin innervation in the territory of the irradiated sciatic nerves was evaluated by immunohistochemistry with neuronal markers. Among these markers, epidermal nerve densities for protein gene product (PGP) 9.5, calcitonin gene-related peptide (CGRP), and substance P (SP) were significantly lower on the irradiated sides than the control sides with a different degree of loss for each marker (42.1-53.1%, P b 0.05). Results suggest that laser-induced focal neuropathy provides a new system for studying neuropathic pain. With this approach, the extent of nerve injury can be quantified. Both smalldiameter epidermal nerves and large-diameter sensory and motor nerves are susceptible to laser-induced injury of different degrees.

[1] 69w Small-diameter sensory nerves of the skin are responsible for conveying noxious and thermal stimuli. Damage to these nerves is presumably related to neuropathic pain behaviors. An understanding of pain mechanisms requires experimental systems to assess the relationship between nerve injury and neuropathic pain and to explore new therapeutic strategies. A critical issue in neuropathic pain studies is to develop an experimental system which produces a quantifiable degree of injury.

[2] 148w Laser irradiation together with photosensitive dyes has become a new approach to induce tissue injury for clinical use, which is known as photodynamic therapy (Dolmans et al., 2003). Only a few reports have employed this approach to induce painful neuropathies (Gazelius et al., 1996;Hao et al., 2000;Kupers et al., 1998). One potential issue is the concomitant requirement of photosensitive dyes. Rosen et al. (2001) showed that laser irradiation alone could elicit damage to endothelial cells of capillaries and small arteries resulting in thrombosis. Previous studies employing laser irradiation indicated that CO 2 laser alone could induce Wallerian degeneration of varying degrees (Menovsky et al., 1996(Menovsky et al., , 2000;;Myers et al., 1985). Those studies did not specify whether neuropathic pain developed in animals after laserinduced nerve injury but raised the possibility that laser irradiation alone under appropriate conditions may produce a new experimental system of inducing neuropathic pain.

[3] 248w Neuropathic pain results in a series of complex but coordinated behaviors mediated by damage to large myelinated, small myelinated, and unmyelinated nerve fibers (Woolf, 2000). We and others have demonstrated the rich innervation of the skin by immunohistochemistry with various neuronal markers, particularly, protein gene product (PGP) 9.5 (Hilliges et al., 1995;Hsieh et al., 2000;Kennedy and Said, 1999;Vaalasti et al., 1988;Wang et al., 1990). PGP 9.5 is a ubiquitin carboxyhydrolase and probably functions as an immediate early gene for processing sensory information in the neurons (Hegde et al., 1997). Our previous study on chronic constriction injury indicates that PGP 9.5(+) nerve terminals in the skin were moderately depleted compared with those in completely denervated skin (Lin et al., 2001). These findings suggest that partial injury is another important principle for creating experimental models of neuropathic pain. An open issue is whether sensory nerve terminals in the skin of different phenotypes, such as nerves positive for calcitonin gene-related peptide (CGRP) and substance P (SP), are depleted to the same degree in neuropathic pain. Apparently, large-diameter and smalldiameter nerve fibers are differentially vulnerable in models of neuropathic pain (Basbaum et al., 1991;Gautron et al., 1990;Guilbaud et al., 1993;Nuytten et al., 1992). Whether terminals of sensory nerves in the skin retain the same patterns as they have at the sciatic nerve level is an open issue. Thus, it was intriguing to investigate whether there is a relationship between nerve injury to fibers of different categories and the magnitude of neuropathic pain.

[4] 44w To address the above issues, we developed a new system of neuropathic pain by laser irradiation alone and took multidisciplinary approaches to evaluate small-diameter sensory nerves by examining the innervation of the skin and large-diameter motor nerves by examining the innervation of motor endplates.

[1] 329w To understand pathophysiological consequences of irradiation with diode laser at 532 nm (100 mW, 30 s) on epineurial vessels of sciatic nerves, we evaluated blood flow and vascular pathology. Before the application of laser, we measured the epineurial blood flow on both sides after removing connective tissues. The appearance of epineurial arteries was smooth (Figs. 1A and B). The blood flows through epineurial vessels on both sides were similar (0.27 F 0.02 mm/s on control side vs. 0.30 F 0.04 mm/s on operated side, P = 0.53; Fig. 1D, E). Three minutes after laser irradiation, the outline of epineurial vessels on the operated side became irregular, and the diameter was reduced (Fig. 1C). The blood flow through the irradiated segment of sciatic nerves was significantly reduced after laser irradiation compared with that before laser irradiation (0.05 F 0.01 mm/s vs. 0.30 F 0.04 mm/s, P = 0.0025, Fig. 1E, F). These blood flow data were supported by ultrastructural evidence of thrombosis in epineurial vessels. Immediately after laser irradiation, thrombi were detected in epineurial vessels at the irradiated segment of the sciatic nerve (Fig. 2A). Thrombosis could also be demonstrated in endoneurial vessels immediately beneath the irradiation site (Fig. 2B). In contrast, endoneurial vessels in the more central region of the nerve fascicle were patent. Endothelial cells of the epineurial vessel were severely damaged with formation of vacuoles. The components of thrombi included red blood cells and platelets (Fig. 3A). Immediately after laser irradiation, myelinated and unmyelinated axons appeared normal in the central region of the nerve fascicle away from the subepineurial region (Fig. 3B). For further demonstration that axons of the sciatic nerve were not directly damaged by the laser irradiation, amplitudes of compound muscle action potential (CMAP) of the plantar muscle were assessed before and 30 min after laser irradiation. Amplitudes of CMAP before and 30 min after laser irradiation on the operated side were 2.42 F 0.09 mV versus 2.46 F 0.07 mV, respectively (P = 0.82).

[2] 182w After laser irradiation, rats exhibited significant changes in behaviors, gait, and stance, including guarding of the affected paw from touching the floor. The most pronounced behavioral changes and abnormal walking patterns occurred during PODs 7-14, and gradually disappeared after POD 28. Rats usually elevated the hind paw of the operated side particularly when walking on uneven surfaces beginning from POD 7 (Fig. 4). They shifted their body weight to the control side while sitting or lying. When they placed the affected foot on the floor, only the medial side of the paw touched the floor. When lying down, the operated hind paw was placed over the scrotum or tail most of the time. Sometimes spontaneous shaking of the operated hind paw was noted. Beginning from POD 2, all toes were flexed together, and there was marked plantar flexion of the hind paw during walking. A limping gait gradually developed on POD 7, and the stance phase of the affected hind limb became shortened. Rats usually walked on toes of the irradiated side with the heel and footpads only briefly touching the floor.

[3] 52w Despite the above observations, rats appeared normal and showed no signs of stress, such as struggling and vocalization when brought to the examination rooms for the thermal and mechanical tests. The fur of the animals was well groomed, and there were no trophic changes in the four limbs. No autotomy was detected.

[4] 108w Rats showed significant thermal hyperalgesia beginning from POD 2, which lasted for 42 days (Fig. 5). Differences in withdrawal latencies between the irradiated side and the control side of each rat were used to assess thermal hyperalgesia, with the baseline values clustering around 0 (0.39 F 0.24 s, P = 0.1138, Fig. 5A). After laser irradiation, differences in the withdrawal latencies became negative from POD 2 (À2.95 F 0.41 s, P b 0.001) and reached the most negative value on POD 7 (À3.16 F 0.19 s, P b 0.001). This value remained significantly lower than the baseline value until POD 42 (À1.56 F 0.43 s, P b 0.05).

[5] 109w To quantitatively describe pain-related behaviors during thermal stimulation, we analyzed the behaviors by measuring elevation time (Fig. 5B) and calculating total behavior scores of the affected hind paws (Fig. 5C). Rats always elevated the hind foot transiently. After laser irradiation, rats elevated the affected limb for a longer period after sensing the noxious heat. The hind paw elevation time significantly increased compared to the baseline value starting from POD 4 (6.50 F 1.20 s, P b 0.05), and this value reached a peak on POD 21 (9.15 F 2.84 s, P b 0.05). The elevation time of the operated hind paws returned to the baseline level after POD 35.

[6] 71w In addition, there were robust changes in the reaction to noxious heat after the surgery; animals usually kept the operated hind paw lifted with licking it. The behavior scores increased from POD 2 (1.90 F 0.17, P b 0.05) and reached the highest value on POD 21 (2.43 F 0.31, P b 0.05). Taken together, thermal hyperalgesia lasted for 3 weeks and gradually returned to the baseline level after POD 35.

[7] 80w The laser irradiation injury also induced mechanical allodynia in addition to thermal hyperalgesia (Fig. 6). Mechanical thresholds were nearly identical before surgery, with the logarithm of the threshold ratio around 0 (0.06 F 0.04, P = 0.19). The withdrawal thresholds to mechanical stimuli were significantly reduced starting from POD 4 (À0.44 F 0.08, P b 0.05). Mechanical allodynia was significant up to POD 21 (À0.51 F 0.16, P b 0.05). Withdrawal thresholds returned to the baseline value after POD 28.

[8] 170w To understand the degree of damage to myelinated nerve fibers after laser irradiation, we examined semi-thin sections of the sciatic nerves on POD 7. There were three zones of nerve injuries in the irradiated segment from the subepineurial region to the central part of the nerve fascicle (Fig. 7A). Degenerating axons with debris were scattered in the area immediately beneath the epineurium (* in Fig. 7A, B). Next to this region was an area containing demyelinating axons (** in Fig. 7A, C). An increase in the extracellular space between axons was noted in the region peripheral to the demyelination zone, and some demyelinating axons could still be found in this region (*** in Fig. 7A, D). There was only endoneurial edema or minimal nerve pathology in the area opposite the site of laser irradiation. In the sciatic nerve distal to the irradiation site, the loss of myelinated fibers was observed in one area of the sciatic nerve with intact axons in the other part of the sciatic nerve (Fig. 8).

[9] 98w We further examined structural changes in NMJs of plantar muscles after laser irradiation. Motor unnervation was demonstrated by combined cholinesterase histochemistry and immunohistochemistry with PGP 9.5 (Fig. 11). NMJs on the control side were abundantly innervated by PGP 9.5(+) axons (Fig. 11A), and these axons extended into the entire NMJ (Fig. 11B). On POD 7 of laser irradiation, some NMJs of the plantar muscle on the operated side became denervated. NMJs at some areas of the plantar muscle were innervated normally (Figs. 11C-D), but NMJs at other parts of the muscle were denervated with axonal debris (Figs. 11E-F).

[10] 57w The ratios of innervated NMJs on both sides were calculated for quantifying the degree of damage to motor nerve terminals. This ratio on the operated side on POD 7 was significantly reduced compared to that on the control side (71.2% F 1.7% vs. 93.3% F 2.0%, P = 0.0002), about 76.3% of the control value (Fig. 12).

[11] 117w To evaluate the damage to small-diameter sensory nerve terminals in the skin, we performed immunohistochemical analyses on footpads of the hind paw innervated by the sciatic nerve and quantified epidermal nerve densities. The epidermis of the control side was abundantly innervated by PGP 9.5(+) fibers (Fig. 13A). These nerves originated from the subepidermal nerve plexus, penetrated through the epidermal-dermal junction and traveled perpendic-ularly in the epidermis. The epidermal nerve density was 10.28 F 0.73 fibers/mm on the control side (Fig. 14A). On POD 7, the PGP 9.5(+) fibers on the operated side were significantly reduced (Fig. 13B), with an epidermal nerve density of 5.46 F 1.63 fibers/mm, 53.11% of the control side (P b 0.05) (Fig. 14A).

[12] 131w Similar to PGP 9.5(+) fibers, CGRP(+) fibers and SP(+) fibers were found in the epidermis of the control side (Figs. 13C, E) with epidermal nerve densities of 4.42 F 0.40 and 2.14 F 0.24 fibers/mm, respectively (Figs. 14B, C). On POD 7 after laser irradiation, CGRP(+) fibers on the operated side were reduced to 2.20 F 0.61 fibers/mm, (P b 0.05), 49.77% of the control side (Figs. 13D, 14B). SP(+) fibers on the operated side had decreased to 0.90 F 0.42 fibers/mm (P b 0.05), 42.06% of the control side (Figs. 13F, 14C). The difference in epidermal nerve density of GAP 43(+) fibers between the operated side and the control side was not statistically significant (5.75 F 1.15 vs. 5.84 F 0.78 fibers/mm, P = 0.22) (Figs. 13G, H, and 14D).

[1] 58w The experiments were performed on adult male Sprague-Dawley rats weighing 200-250 g. Animals were housed in plastic cages whose floors were covered with sawdust to avoid mechanical damage to the hind paw skin. Sufficient water and food were provided. The experiments followed the guidelines of the International Association for the Study of Pain (IASP) (IASP Committee, 1980;Zimmermann, 1983).

[2] 61w Two measures were employed to assess thermal hyperalgesia and mechanical allodynia after irradiation (Chaplan et al., 1994;Hargreaves et al., 1988). Rats were adapted to the test environment for 5-7 days before testing. The baseline responses were recorded before irradiation of the sciatic nerve. After surgery, rats were tested on days 2, 4, and 7 and then weekly during the experimental period.

[3] 173w To evaluate the response to thermal stimulation, rats were assessed with the paw-withdrawal test of the Hargreaves type (Ugo Basile, Comerio, Italy) (Hargreaves et al., 1988;Lin et al., 1997). Animals were placed in a plastic box on a glass plate. The plantar surface of the hind paw was directly stimulated with an infrared source through the glass plate. Two parameters were evaluated: (1) the paw-withdrawal latency, defined as the interval between the onset of heat stimulation and withdrawal of the hind paw, and (2) the hind paw elevation time, defined as the interval between withdrawal of the hind paw and replacement of the paw on the floor. Both parameters were measured to the nearest 0.1 s. The hind paws were tested in a random fashion. Each paw was tested five times with a 5-min interval between consecutive trials. The five withdrawal latencies per side were averaged. The difference between the two sides (the operated side minus the control side) was the withdrawal latency difference, with a value of z2 s defined as hyperalgesia.

[4] 71w During the five tests, the intensity of the withdrawal responses was further categorized as 1 point for withdrawal with a brief pawlift, 2 points for a short-interval withdrawal (V5 s) sometimes with a transient paw-lick, and 3 points for prolonged withdrawal (N5 s) with vocalization, escaping, and prolonged licking of the tested paw, and sometimes with gentle biting. The weighted mean of the five responses was defined as the behavioral score.

[5] 178w To examine the response to mechanical stimulation, a set of 17 calibrated von Frey hairs (0.026 -110 g, Somedic, Sweden) was used for assessment (Chaplan et al., 1994). Animals were placed in a plastic box on a metal mesh floor with a habituation period of 10 min. The testing was initiated with the hair weighing 3.30 g, and each foot was examined in a consecutive fashion with a descending or ascending hair number according to the response. Five stimuli using the selected hair were applied at 5-s intervals. If there was no withdrawal response to the initially selected hair with these five stimuli, a stronger stimulus was applied. If the animal withdrew its hind paw in response to any of the five stimuli, the next weaker stimulus was chosen. The mechanical threshold was expressed as the minimal force ( g) initiating a withdrawal response. The results were expressed as the logarithm of the withdrawal ratio (operated side over control side). Negative data indicated a reduction in the mechanical threshold of the operated side compared to the control side.

[6] 154w The motor function of the sciatic nerve was assessed weekly after the operation (Ko et al., 1999). Rats were anesthetized before evaluation, and the compound muscle action potential (CMAP) was measured with an evoked response recorder (Neuropack II, MEB-5100, Nihon Kohden, Tokyo, Japan). The stimulating electrodes were inserted and placed at the sciatic notch to stimulate the sciatic nerve, and the recording electrodes were on the plantar muscles. Amplitudes of the CMAP on both sides were recorded for analysis. Sensory nerve action potential (SNAP) of the sural nerve was recorded orthodromically. The sural nerve was exposed and gently dissected free from the surrounding connective tissues in the popliteal fossa. The stimulating surface electrodes were placed at the lateral side of the foot dorsum, distal to the lateral malleolus. The needle recording electrodes were placed beside the sural nerve near the sciatic nerve trifurcation. Amplitudes of the SNAP on both sides were recorded for analysis.

[7] 253w For immunohistochemistry on frozen microtome sections (Lin et al., 2001), animals were fixed with an intracardiac perfusion with 4% paraformaldehyde in 0.1 M (PB), pH 7.4. The skin areas innervated by the sciatic nerve were fixed for another 6 h and then changed to PB for storage. After thorough rinsing in PB, samples were cryoprotected with 30% sucrose in PB overnight. Sections perpendicular to the epidermis were cut at 30 Am on a sliding microtome, labeled sequentially, and stored at À208C. To ensure adequate sampling, every fourth section for each tissue was chosen for immunohistochemistry. Sections were treated with 0.5% Triton X-100 in 0.5 M Tris buffer (Tris), pH 7.6, for 30 min and processed for immunostaining. Briefly, sections were quenched with 1% H 2 O 2 in methanol and blocked with 5% normal goat serum in 0.5% nonfat dry milk/Tris. Sections were incubated with rabbit antiserum to PGP 9.5 (UltraClone, Isle of Wight, UK, 1:1000), CGRP (Chemicon, Temecula, CA, 1:2000), SP (DiaSorin, Stillwater, MN, 1:1000), and growth-associated protein 43 (GAP 43) (Chemicon, Stillwater, MN, 1:1000) for 16-24 h. CGRP and SP are neuropeptides related to sensory perception (Ma and Bisby, 2000). GAP 43 is a neuronal presynaptic membrane protein that is generally considered to be a marker of neuronal plasticity (Doubell and Woolf, 1997). After rinsing in Tris, sections were incubated with biotinylated goat antirabbit IgG for 1 h, and the avidin-biotin complex (Vector, Burlingame, CA) for another hour. The reaction product was demonstrated by 3,3V -diaminobenzidine (DAB, Sigma, St. Louis, MO).

[8] 370w In the first phase of the experiment, we investigated the optimal duration of laser irradiation for inducing neuropathic pain. The criterion of successful induction was the presence of sustained thermal hyperalgesia at least in the interval between postoperative days (PODs) 7 and 14, when animals usually showed the maximal neuropathic pain behaviors according to previous studies on different neuropathic pain systems including chronic constriction injury (Bennett and Xie, 1988), partial sciatic nerve ligation (Seltzer et al., 1990), tight ligation of spinal nerves (Chaplan et al., 1994), and spared nerve injury (Decosterd and Woolf, 2000). Clearly, the success rate of inducing neuropathic pain depended on the duration of laser irradiation (Table 1). Irradiation with a power of 100 mW for 30 s induced the highest rate of neuropathic pain induction (four of six animals). Laser irradiation of a shorter duration (15 s) did not produce significant changes in the paw-withdrawal latencies on noxious heat stimulation. A longer duration of laser irradiation (60 s) generated various outcomes; three animals exhibited thermal hyperalgesia, and the others showed thermal anesthesia as reflected in the large variation in paw-withdrawal latencies. We therefore determined that laser irradiation of 100 mW for 30 s was the optimal dose and used this setting to induce neuropathic pain in the animals. In total, there were 62 animals (including the six rats in the firstphase experiment) subjected to laser irradiation of 100 mW for 30 s, and 37 of them with an overall success rate of 59.7% fulfilled the criteria described above. These 37 animals were used for characterization of behavioral, electrophysiological, and pathological changes. At each time point, there were at least five animals for the laboratory procedures (semi-thin sections and immunohistochemistry of footpads). All procedures of measurement, quantitation, and analysis were performed in a blinded fashion. Behavioral and laboratory data were presented as the mean F SEM at different time points after laser irradiation. For statistical analysis of values obtained from behavior testing over the experimental period, repeated-measures ANOVA followed by Dunn's post hoc test was used. Differences in the values in amplitude of CMAP and epidermal nerve density between control and operated sides were tested using Student t test. Values of P b 0.05 were considered significant.

[9] 222w This report documents a new experimental pain-inducing system of focal neuropathy by using brief laser irradiation alone without photosensitive dyes. The present study demonstrates that the nerve injury produced by such a simple approach can induce neuropathic pain in addition to laser-induced ischemia (Rosen et al., 2001). Characteristic manifestations include spontaneous and nociception-evoked pain behaviors, thermal hyperalgesia, and mechanical allodynia similar to other neuropathic pain systems induced by mechanical injury as summarized in Table 2. There are certain unique features in the current approach compared with photochemical systems. First, the durations of neuropathic pain, including mechanical allodynia and thermal hyperalgesia, were shorter than with the other types; thermal hyperalgesia lasted for 5 weeks in the current system, compared to 2-3 months with the photochemical approach (Hao et al., 2000;Kupers et al., 1998) and mechanical injury (Bennett and Xie, 1988;Kupers et al., 1992;Malmberg and Basbaum, 1998). Several factors may account for these differences. For example, in the current approach, the effect of laser irradiation was limited to one portion of the sciatic nerve fascicle, particularly, the subepineurial regions immediately underneath the site of laser irradiation. Other alternatives may include potential reversibility of nerve lesions or less extensive changes in central sensitization (Craig, 2003); confirming these possibilities requires further investigations. Nevertheless, this approach provides a simple and brief procedure to generate focally painful neuropathy.

[1] 193w Rats were anesthetized by an intraperitoneal injection of chloral hydrate (400 mg/kg). The sciatic nerve was exposed at the midthigh level after a dorsolateral skin incision and splitting of the fascia between the gluteus and biceps femoris muscle. The nerve was gently dissected from the surrounding connective tissues over a distance from the gluteus muscle to the trifurcation of the sciatic nerve. The segment of the sciatic nerve just distal to the gluteus muscle was marked with epineurial sutures and irradiated for various periods under a laser beam. The source of laser irradiations came from a diode-pumped solid state laser operating at 532 nm (HCP Corp., Hsinchu, Taiwan) with an output power of 100 mW. The beam of the laser (1-mm diameter) was focused on the epineurial vessels of the sciatic nerve. For each animal, the operation was performed on one side, with the other side undergoing a sham operation. The intensity of the laser output was measured with a laser power meter (LaserCheck, 33-1553, Coherent, Auburn, CA) before and after the surgery. Sham operation was performed on control sides with similar procedures of removing connective tissues except that laser irradiation was omitted.

[2] 147w Epineurial blood flow of sciatic nerves was measured with a laser Doppler flowmeter (CAM1, KK Research Technology, Devon, England) before and immediately after laser irradiation (Morris et al., 1996). The sciatic nerve was exposed at the thigh level and covered with a pool of paraffin oil to prevent dryness of the nerve. A laser Doppler probe with a spot of 10 Am in diameter was positioned perpendicularly to the nerve segment covered by a drop (approximately 50 Al) of liquid paraffin oil. This minimal amount of paraffin oil only covered the surface of the sciatic 7 nerve and did not interfere with the measurement of blood flow. During the 10-min examining period, no more paraffin oil was added. Before and after laser irradiation, blood flow data were collected for an interval of 2 min, respectively. The surface of the sciatic nerves was still wet after the test.

[3] 136w Animals were perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, and the irradiated and distal parts of the sciatic nerve were postfixed in 5% glutaraldehyde in 0.1 M PB overnight (Lin et al., 1997). Tissue was postfixed in 2% osmic acid for 2 h at room temperature, dehydrated with a graded series of alcohol, and embedded in Epon 812 resin (Polyscience, Philadelphia, PA). Cross-sections of 1 Am were cut on an ultramicrotome, dried on slides using a hot plate, stained with toluidine blue, and observed under a light microscope. Axonal degeneration, demyelination, and the degree of endoneurial edema were evaluated according to established criteria (Iida et al., 2003;Nakuda et al., 2002). Selected areas were thin-sectioned, doubly stained with uranyl acetate and lead citrate, observed under a Hitachi electron microscope, and photographed.

[4] 142w Epidermal innervation was quantified according to modified protocols in a coded fashion (Hsieh et al., 2000). PGP 9.5-, CGRP-, SP-, and GAP 43-immunoreactive nerves in the epidermis of each footpad were counted at a magnification of Â400 with an Olympus BX40 microscope (Tokyo, Japan). Each individual nerve with branching points inside the epidermis was counted as one. For epidermal nerves with branching points in the dermis, each individual nerve was counted separately. The total length of the epidermis along the upper margin of the stratum corneum in each footpad was measured using the Image-Pro PLUS system (Media Cybernetics, Silver Spring, MD). Epidermal nerve density was therefore derived and expressed as the number of fibers per millimeter of epidermal length. Every fourth section of each tissue was quantified, and there were three sections for each footpad. All slides were coded during the quantitation.

[5] 103w For morphological examination of the motor innervation, cholinesterase histochemistry combined with immunohistochemistry was performed on the plantar muscles following established protocols (Ko et al., 1999). The plantar muscles were dissected after overnight postfixation. Serial 30-Am cryostat sections were mounted on gelatin-coated slides. Every fifth section was stained with cholinesterase histochemistry and immunohistochemistry for PGP 9.5. This method demonstrates the neuromuscular junctions (NMJs) and motor innervation simultaneously. Coded sections from the control side and the operated side were observed at a magnification of Â400 under an Olympus BX40 microscope. The ratios of innervated NMJs to total NMJs on each section were calculated for analysis.

[6] 68w To investigate the effect of motor nerve damage after laser irradiation, we measured the amplitudes of CMAPs of the sciatic nerves. There was significant reduction in the amplitudes of CMAPs after laser irradiation (Fig. 9A). Before surgery, the amplitude of CMAP was 2.46 F 0.04 mV. On POD 7, this value was reduced to 1.60 F 0.09 mV, 65.0% of the control side (Fig. 9B) (P b 0.0001).

[7] 63w To evaluate the damage to large-diameter sensory nerves, amplitudes of SNAPs of the sural nerves were recorded on POD 7 (Fig. 10A). The amplitude of SNAP on the operated side was significantly reduced compared to that on the control side (71.00 F 15.55 AV vs. 167.10 F 17.99 AV, P = 0.0043, Fig. 10B), about 42.5% of the value on the control side.

[8] 247w Nerve injury is an essential factor in inducing neuropathic pain, and an important issue for elucidating different mechanisms of neuropathic pain requires an animal system of neuropathic pain with quantifiable nerve injury. The present system indicates that the duration of laser irradiation could be adjusted to induce neuropathic pain. Laser irradiation of 15 s produced the lowest success rate of neuropathic pain lasting for only 1 week. In contrast, laser irradiation for 60 s resulted in a combination of thermal hyperalgesia and anesthesia. The result extends previous studies on freezing injury-induced neuropathic pain, in which the magnitude and duration of the hyperalgesia were related to the extent of nerve damage (Myers et al., 1996;Wagner et al., 1995). Several lines of evidence have indicated that the injured area in the sciatic nerve is proportional to the magnitude of the insults applied to the nerve. In studies using the CO 2 laser, the stronger the laser energy used to irradiate the sciatic nerve, the greater nerve damage that was noted (Menovsky et al., 1996;Myers et al., 1985). However, it has not been demonstrated that these CO 2 laserinduced nerve injuries could cause neuropathic pain (Menovsky et al., 1996(Menovsky et al., , 2000;;Myers et al., 1985). Thus the dose of laser energy is an adjustable factor, so that the degree of nerve injury and the extent of neuropathic pain behaviors can be predicted. The current approach therefore is useful in determining relationships among nerve injury, pain behaviors, and underlying mechanisms.

[9] 132w Laser-induced nerve injury can be classified into two phases: an immediate phase and a late phase. The immediate thermal effect of laser irradiation in the current report was limited to the subepineurial region and depended on the nature and dose of the laser. This is in contrast to the CO 2 laser, which causes much more extensive thermal injury than the diode laser of 532 nm used in the current system (Menovsky et al., 1996(Menovsky et al., , 2000)); after CO 2 laser irradiation on sciatic nerves, both the epineurium and endoneurium are severely damaged by the thermal effect. The entire nerve becomes edematous, and pyknotic nuclei appear in fibroblasts. All these signs indicate extensive injury by the CO 2 laser, which is absorbed by tissue water and causes vaporization of tissue.

[10] 101w Subsequent injury was related to ischemic events as demonstrated by the formation of thrombi and reduced blood flow through epineurial vessels. Based on previous studies comparing tight ligation and chronic constriction injury, it is possible that extensive injury to the sciatic nerve will cause anesthesia instead of hyperalgesia (Lin et al., 2001). In the current system, only a limited portion of the subepineurial area was injured by laser irradiation during the acute stage, while other portions of the sciatic nerves remained intact microscopically. This approach provides an opportunity to investigate the degree of nerve injury and the occurrence of neuropathic pain.

[11] 404w The vulnerability of large-diameter versus small-diameter nerves in painful neuropathy is an intriguing issue (Basbaum et al., 1991;Gautron et al., 1990;Guilbaud et al., 1993;Nuytten et al., 1992); this issue is complicated because ischemic insults and mechanical injuries can generate various outcomes (Parry and Brown, 1982;Vital et al., 1986). The extent of nerve damage by laser irradiation appears differentially, including large-diameter versus small-diameter nerve fibers, and different phenotypes of sensory nerve terminals in the skin. Epidermal nerves of PGP 9.5, CGRP, and SP phenotypes were reduced more prominently (42.1-53.1% of the control side) compared to large-diameter motor nerves as evaluated by the ratios of innervated NMJs (76.3% of the control side). This finding indicates that the damage to smalldiameter nerves was more extensive than that to large-diameter nerves in the laser-induced focal neuropathy. This is in contrast to other neuropathic pain models using the photochemical approach; myelinated axons were much more susceptible than unmyelinated axons to damage (Hao et al., 2000;Kupers et al., 1998). Differences in the extent and degree of nerve injury may underlie the difference. Alternatively, different spatial patterns of nerve fibers may contribute to the differential degrees of large-versus small-fiber damage. Intriguingly, epidermal nerve fibers of various phenotypes are differentially vulnerable to laser irradiation. On human studies, skin innervation is reduced in neuropathic pain, such as painful sensory neuropathy (Holland et al., 1997;Periquet et al., 1999) and postherpetic neuralgia (Oaklander et al., 1998). In animal models of neuropathic pain, several groups including ours have demonstrated a reduction of epidermal nerve densities after partial sciatic nerve injury and chronic constriction injury (Lin et al., 2001;Lindenlaub and Sommer, 2002;Ma and Bisby, 2000). It is not clear whether different subtypes of epidermal nerves are reduced to the same extent. The reduction of epidermal nerves in the current model not only extends previous observations of skin denervation with neuropathic pain, but also provides additional information regarding the susceptibility of epidermal nerves of different phenotypes to laser irradiation. The reduction in GAP 43(+) nerves was minimal, while epidermal nerves positive for PGP 9.5, CGRP, and SP were reduced with laser-induced neuropathic pain. This is in contrast to mechanical injury, in which epidermal nerves of different phenotypes were damaged to a similar degree (Lin et al., 2001;Lindenlaub and Sommer, 2002). These results suggest that partial injury is required to produce neuropathic pain and that epidermal nerves of different phenotypes are susceptible to different types of neuropathic pain models.