J Mol Hist (2007) 38:245–251 DOI 10.1007/s10735-007-9094-5
ORIGINAL PAPER
Microglial activation in different models of peripheral nerve injury of the rat ˇ ´ızˇkova´ Stanislava Jergova´ Æ Da´sˇa C
Received: 20 November 2006 / Accepted: 18 April 2007 / Published online: 15 May 2007 Springer Science+Business Media B.V. 2007
Abstract Pain and pain modulation has been viewed as being mediated entirely by neurons. However, new research implicates spinal cord glia as key players in the creation and maintenance of pathological pain. Sciatic nerve lesions are one of the most commonly studied painrelated injuries. In our study we aimed to characterize changes in microglial activation in the rat spinal cord after axotomy and chronic constriction injury of the sciatic nerve and to evaluate this activation in regard to pain behavior in injured and control groups of rats. Microglial activation was observed at ipsilateral side of lumbar spinal cord in all experimental groups. There were slight differences in the level and extent of microglial activation between nerve injury models used, however, differences were clear between nerve-injured and sham animals in accordance with different level of pain behavior in these groups. It is known that activated microglia release various chemical mediators that can excite pain-responsive neurons. Robust microglial activation observed in present study could therefore contribute to pathological pain states observed following nerve injury. Keywords Peripheral nerve injury Neuropathic pain Allodynia Microglia
Introduction A number of rat peripheral neuropathy models have been developed to simulate human neuropathic pain conditions. S. Jergova´ (&) D. Cˇ´ızˇkova´ Institute of Neurobiology, Slovak Academy of Sciences, 04001 Kosice, Slovak Republic e-mail:
[email protected]
Sciatic nerve lesions are one of the most commonly studied injuries. The most obvious form of neuropathy is complete transection of the peripheral nerve, where neuroma at the site of injury and dorsal root ganglion proximal to the trauma are the source of spontaneous ectopic activity, resulted in development of abnormal pain sensations (Wall and Devor 1983). A commonly used partial nerve injury technique is the chronic constriction injury (CCI), developed in rat by Bennett and Xie (Bennett and Xie 1988). In this model the sciatic nerve is loosely ligated with four chromic gut sutures and animals develop spontaneous painrelated behavior, allodynia and hyperalgesia to thermal and mechanical stimuli. Significance of the role of glial cells in the pathology of the central nervous system (CNS) is changing in recent years. Glial cells were thought to be passive cells with weak responses to synaptic activation (Haydon 2001). However, growing body of evidences implicates spinal cord glia not only as supporting cells for CNS neurons but also as an important modulator of neuronal functions under physiological and pathophysiological conditions (Watkins et al. 2001a; Watkins et al. 2001b). Glial cells are well positioned to influence neuronal functioning as they encapsulating neurons and express receptors for many neurotransmitters and transporters (Bruce-Keller 1999; Kommers et al. 1998; Palma et al. 1997). Microglia represent 5–10% of glia in CNS (Kreutzberg 1996; Stoll and Jander 1999). In adults, microglia are distributed throughout CNS and have a small soma bearing thin and branched processes under normal conditions. Microglial cells act as sensors for a range of stimuli that threaten physiological homeostasis, including CNS trauma, ischemia and infection. Activation of microglia by one or more of these stimuli results in progressive series of changes in microglial cells morphology, gene expression,
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function and number. Activated microglia produce and release various chemical mediators, including proinflammatory cytokines that can produce immunological actions (Hanisch 2002; Kreutzberg 1996; Nakajima and Kohsaka 2001; Stoll and Jander 1999). Activated glial cells also release substances that excite spinal pain-responsive neurons, such as reactive oxygen species, nitric oxide, prostaglandins, grow factors and other (Hanisch 2002; Woolf and Salter 2000). Moreover, these substances caused release of ‘‘pain’’ transmitters from sensory neurons in the dorsal horn (Inoue et al. 1999; Marriott 2004; Rasley et al. 2002). The aim of this study was to evaluate possible changes in microglia activation in different models of peripheral nerve injury at the level of spinal cord and to evaluate this activation in regard to different level of pain behavior between injured and sham groups of rats.
Materials and methods Experiments were done in accordance with the regulations of the Animal Care and Use Committee at the Institute of Neurobiology, Slovak Academy of Sciences and following the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmermann 1983). All animals were housed in groups of 2 or 3 in clear plastic cages, exposed to light 12 h per day. Food and water were available ad libitum. Surgical procedure Experiments were performed on adult male Wistar rats (300–350 g). Animals were divided into three groups; animals in ‘‘CCI group’’ (n = 6) received unilateral chronic constriction injury of the sciatic nerve, animals in ‘‘transection group’’ (n = 6) received unilateral transection of the sciatic nerve, animals in ‘‘sham group’’ (n = 4) received unilateral sham operation. Peripheral neuropathy was induced under halothane anesthesia (2% for induction, 0.8% for maintenance) in a 1:1 mixture O2 and N2O. The common sciatic nerve was exposed at mid-thigh level and (i) tightly ligated with two nylon sutures and transected between the pair of ligatures (ii) four ligatures (4.0 chromic gut) were loosely tied with about 1 mm spacing proximally to the sciatic trifurcation. The nerve was then placed to its original location. An identical exposure was performed in the sham-operated animals, but no axotomy/ligation was done. After complete hemostasis was confirmed the wound was closed in layers, using vicryl 3-0 for muscle and nylon 3-0 for skin. Animals were left to survive for 7 days.
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Behavioral test Behavioral tests were performed at room temperature between 10:00 a.m. and 1:00 p.m. The presence of tactile allodynia was performed using a series of von Frey filaments with a logarithmically incremental stiffness as described in previous studies (Chaplan et al. 1994). Rats were transferred to a clear plastic testing chamber with a wire mesh bottom (18 cm · 25 cm) and allowed to acclimatize for 20 min. Measurements on both ipsilateral and contralateral sides in nerve-injured as well as in sham operated animals were performed in the medial third of the hind paw. The filaments in range 0.6–15 g were applied, in consecutive sequence, to the plantar surface of the hind paw pads on the ipsilateral/contralateral side of the nerveinjured and sham-operated rats. A filament of buckling weight 2 g was used as first. A vertical elevation of the paw immediately upon removal of the testing hair was considered to be a positive response and filament was replaced by weaker one. When negative response appeared the next stronger filament was used. The scores obtained from this procedure were used to calculate a 50% paw withdrawal threshold (PWT) using the up-down method (Dixon 1980). Imunohistochemistry After a survival period rats were deeply anaesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.) and perfused intracardially with saline followed by 4.0% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH = 7.4, Sigma). Spinal cord segments L4-L5 were removed, postfixed in the same fixative for 12 h at 4C and then transferred to a PBS containing sucrose (15–20%). The next day segments were serially sectioned at 30 lm thickness in a transverse plane with a freezing microtome. Free-floating sections were stained using a standard avidinbiotin-peroxidase complex (ABC) technique. Sections were incubated overnight in the primary rabbit antiserum directed against Iba-1 protein (Wako, 1:3,000) in 0.1 M PBS containing 5% normal goat serum (NGS), followed by incubation in biotinylated goat antirabbit IgG, diluted 1:200 in PBS and 5% NGS and then reacted with ABC (Vectastain, Vector Lab.) The reaction product was visualized with 0.03% hydrogen peroxide and 0.05% 3,3¢diaminobenzidine-tetrahydrochloride solution as the chromogen. Sections were then mounted on slides, air-dried, dehydrated through graded ethanol solutions followed by xylene and then coverslipped with Permount. Sections were viewed with bright field microscope Olympus BX51/ BX52 and digitalized with an Olympus DP50 digital camera coupled with computer equipped with Olympus DP Image software (version 3.1).
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Quantification of immunolabeling For quantification of Iba-1 immunoreactivity (Iba-1-IR) ten sections of L4-L5 spinal cord segments were taken from each nerve-injured and sham-operated rat. Obtained photomicrographs of selected spinal cord sections were exported to UTHSCSA Image Tool Program where density of Iba-1-IR was determined by the experimenter unaware of treatment group. Density was measured in the dorsal horn and in lamina IX of the ventral horn. For each section density of background was subtracted from density of Iba-1 immunostaining. For statistical evaluation of differences in density of staining between contra and ipsilateral sides of the spinal cord sections a paired comparison using a Student t-test was performed. One-way analysis of variance (ANOVA) was used to evaluate differences between CCI and sham group. Data are expressed as mean ± SEM.
Results von Frey stimulation Sensitivity to light mechanical stimuli was tested with von Frey filaments. During one week before surgery all animals were tested for baseline paw withdrawal threshold (PWT) that was about 13 g (PWT = 12.7 ± 1.3 g). Significant decrease of PWT to von Frey stimulation was observed in animals with injured sciatic nerve when compared to sham animals during first week following injury. No significant differences were observed between groups of animals with injured nerve. These animals developed mechanical allodynia since day 3 following injury. PWT at this time point was 6.28 ± 1.3 g and 4.11 ± 1.5 g for ipsilateral hind paw of CCI animals and animals with transected nerve respectively. At the end of the week ipsilateral hind paw PWT level decreased to 3.28 ± 1.8 g and 1.95 ± 1.7 g for CCI and axotomy group respectively. PWT of sham animals remained almost unchanged (Fig. 1). Iba-1 immunostaining Sham injury resulted in mild microglial activation at the central part of ipsilateral dorsal horn laminae I-II of lumbar spinal cord where higher level of Iba-1-IR was presented when compared to contralateral side and morphological changes of activated microglial cells were also visible. Both sciatic nerve lesions produce robust microglial activation when compared to sham injury group (Fig. 2). There were only slight differences in the level of Iba-1-IR between axotomy and CCI model, as axotomy appears to produce more intense microglial activation.
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The localization of the most abundant activated microglial cells was similar in both nerve injury groups: intense Iba-1-IR was observed in the entire region from central part of superficial dorsal horn laminae to medial part of the base of the dorsal horn (lamina VI). In the ventral horn, microglial activation was restricted to lateral part of lamina IX consisted of motoneurons, reflecting injury to motoric fibers (Fig. 3). Activated microglia were characterized by oval amoeboid soma with a few short processes in contrast to highly ramified resting microglia observed at contralateral side of the spinal cord in experimental animals (Fig. 4).
Discussion Calcium-binding protein Iba-1 is highly expressed in cells of the monocyte/macrophage lineage and it is upregulated in activated microglial cells. Expression if Iba-1 was never observed in neurons or astrocytes, therefore this antibody is valuable for identification of microglia and macrophages (Imai and Kohsaka 2002). In our study we followed changes in the level of expression of Iba-1 protein one week following injury of the sciatic nerve. We used chronic constriction injury of the sciatic nerve and transection of this nerve as models of neuropathic pain. In both models animals developed allodynia to mechanical stimuli with significant reduction of paw withdrawal threshold one week following nerve injury. Microglial activation was observed at this time at ipsilateral dorsal horn of the injured animals with weak changes on contralateral sides or in sham animals. Although the reaction of glia to peripheral nerve injuries has already been shown in many experiments, the role of glial cells in the development and maintaining of chronic pain states that often accompanying nerve injury is still the matter of discussion. The role of glia in exaggerated pain states has been first considered by Garrison et al. (Garrison et al. 1994). They used CCI model of pain to study changes in astrocytes activation and found out that pharmacological block of astrocytes activation caused alleviation of exaggerated pain states. The involvement of microglial population in pain states has been suggested from experiments where treatment with minocycline, the inhibitor of microglial activation led to decrease of thermal hyperalgesia and tactile allodynia induced by nerve injury (Tikka et al. 2001; Tikka and Koistinaho 2001). However, several studies showed that minocycline failed to reverse chronic pain (Ledeboer et al. 2005; Raghavendra et al. 2003). In these studies minocycline was able to inhibit microglial activation while had no effect on the activity of astrocytes. These results suggest
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Fig. 1 Paw withdrawal threshold of ipsilateral hind paws of experimental animals during 7 days following injury of the sciatic nerve. Significant decrease of PWT on day 3 following CCI and transection injury indicate development of mechanical allodynia. Further PWT decrease was observed also at day 7 following injury. Development of mechanical allodynia was similar between CCI and transection injury. Data are presented as mean ± SEM, #P < 0.05, ## P < 0.01 for injury versus sham (t-test)
Fig. 2 Density of Iba-1-IR in the spinal cord of sham, CCI and transected nerve animals in dorsal horn and L IX of ventral horn. Iba1-IR was higher at ipsilateral side of the dorsal horn when compared to contralateral side in all groups of animals. Increase of Iba-1density in lamina IX of the ventral horn was observed only in animals with nerve injury. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01 for ipsilateral versus contralateral side, #P < 0.05, ## P < 0.01 for group comparison (ANOVA)
that microglia and astrocytes could play diverse roles in development and maintenance of pain-states. Separate roles of microglia and astrocytes has been studied by Colburn et al. (Colburn et al. 1997; Colburn et al. 1999) in experimental models of peripheral nerve injury. In their experiments peripheral nerve injury led to increase of the astrocyte reaction while very weak changes have been observed in the microglial reaction. Moreover, the reaction of microglial cells was very similar comparing ipsilateral and contralateral sides of the spinal cord sections. Our results seems to be contradictory as we show that chronic constriction injury of the peripheral nerve caused very intensive increase in the reaction of microglia with significant differences from control rats as well as with different reaction in the ipsilateral and contralateral sides of the appropriate spinal cord sections of injured animals. The reason of such discrepancy in results could be in a different
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Fig. 3 Iba-1 immunoreactivity in spinal cord sections of animals seven days following sham injury (A), CCI (B) and transection (C) of the sciatic nerve. Strong microglial activation was observed at ipsilateral sides of spinal cord sections of animals with injured sciatic nerve
degree of sciatic nerve injury or in a different kind of antibody used to identify microglial cells. However, in the experiment of Zhang and Koninck (2006) and Stuesse et al. (2000) the reaction of microglial cells following CCI was similar as in our experiment although they use OX-42 antibody for identification of activated microglial cells. Moreover, microglial reaction in our experiment correlated with the degree of mechanical allodynia observed in the injured animals. It is known that the reaction of spinal glial cells is dependent on the type and site of nerve injury (Colburn et al. 1999). Several studies have shown that activation of microglial cells do not correlate with the
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Fig. 4 Detailed image of Iba-1 positive cells at ipsilateral (A) and contralateral (B) dorsal horn of CCI rat. Changes in morphology of activated microglial cells are obvious when comparing level of ramification between cells on ipsilateral and contralateral side
development and maintenance of pain behavior and more important role for chronic pain states has been suggested for astrocytes (Colburn et al. 1999; Hashizume et al. 2000; Raghavendra et al. 2003; Winkelstein and DeLeo 2002) However, recent experiments by Narita et al. (Narita et al. 2006) showed, that intrathecal application of activated astrocytes did not caused changes in the latency of paw withdrawal to thermal stimulus. Decrease in PWT has been observed only after application of activated microglial cells. These results suggest that astroglial activation is not directly involved in development of pain-states and that activated microglial cells seem to be essential for development of neuropathic pain states following nerve injury. Sciatic nerve injuries used in our experiments caused intense upregulation in Iba-1 protein at ipsilateral side of spinal cord dorsal horn in its central and medial part, i.e. in the area where nociceptive fibers terminate (Willis and Coggeshall 1991). It is known that nociceptive nerve terminals released substances such as substance P, excitatory aminoacids, adenosine triphosphate, nitric oxide and prostaglandins that excite spinal cord neurons and also activated glia (Bezzi et al. 1998; Hide et al. 2000; Marriott et al. 1991; Molina-Holgado et al. 1995). However, activation of glial cell can be induced by pain-responsive neurons themselves. This neuron-to-glia communication is suggested to be mediated by fractalkine expressed on the extracellular surface of neurons (Hatori et al. 2002; Verge
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et al. 2004). Previous studies have shown that spinal cord neurons responding to pain stimuli in this model of neuropathic pain are localized mainly in the superficial laminae I-II and in deeper dorsal horn laminae (Munglani et al. 1999; Ro et al. 2004; Yamazaki et al. 2001). Activation of microglial cells observed in our experiment was localized in the corresponding areas. In both model of peripheral nerve injury we also observed activation of microglial cells in the ventral horn lamina IX. Such reaction is commonly observed around axotomized motoneurons. Ensheathing of motoneurons by microglial cells has presumably neuroprotective effect as it is necessary for displacement of synapses and reducing of excitatory input following axotomy (Blinzinger and Kreutzberg 1968; Streit 2002). Activated microglial cells change their morphology from a resting, ramified shape into an active, amoeboid shape. These morphological differences were obvious when comparing ipsilateral and contralateral sides of dorsal horns of nerve-injured animals. Amoeboid shaped Iba-1-IR cell with short processes were presented in the centromedial area of the ipsilateral dorsal horn while highly ramified microglia were in the contralateral dorsal horn. However, peripheral nerve injury caused not only the morphological changes of microglial cells but could led to proliferation of them. Significant increase of 5-bromo-2deoxyuridine, cell proliferation marker has been observed recently in a model of CCI in mice (Narita et al. 2006). Differences in the extend and intensity of microglial activation between sham and nerve- injured animals were in agreement with clear pain-related behavior observed only in groups with nerve injury. In our previous studies we followed the changes in the c-Fos protein and neural nitric oxide synthase immunoreactivity at the spinal cord and DRG levels after sciatic nerve injury in comparison with development of hyperalgesic pain states, however, we did not detect correlation in the induction or changes in the level of these substances in nerve-injured animals with the increase of hyperalgesia one week after nerve injury (Cizkova et al. 2002; Jergova and Cizkova 2005). Robust microglial activation observed in present study could contribute to pathological pain states in sciatic nerve injury models. Our results support the role of microglia in the maintaining of pain following peripheral nerve injury. As neuropathic pain is by large not relieved by current therapies that target neurons, it is suggested that targeting glia might provide a novel approach for treatment of such unremitting pain conditions. Acknowledgements The authors thank to M. Spontakova for excellent technical assistance. This work was supported by Science and Technology Assistance Agency under the contract No. APVT-51-
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250 011604 and by grant of Slovak Academy of Sciences VEGA No. 2/ 5136/25.
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