Sylvie Multon

Systemic nitroglycerin increases nNOS levels in rat trigeminal nucleus caudalis.

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraineurs a delayed attack of unknown mechanisms. Subcutaneous nitroglycerin (10 mg/kg) produced a significant increase of nitric oxide synthase (NOS)- and c-fos-immunoreactive neurons in the cervical part of trigeminal nucleus caudalis in rats after 4 h. This effect was not observed in the thoracic dorsal horn. Similar increase of NOS and c-fos was obtained in the brain stem after a somatic nociceptive stimulus, i.e. on the side of the formalin injection in the lip. Nitric oxide is thus able to increase NOS availability in second order nociceptive trigeminal neurons, which may be relevant for central sensitization and the understanding of its effect in migraine.

Delayed GM-CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages

Macrophages (monocytes/microglia) could play a critical role in central nervous system repair. We have previously found a synchronism between the regression of spontaneous axonal regeneration and the deactivation of macrophages 3–4 wk after a compression‐injury of rat spinal cord. To explore whether reactivation of endogenous macrophages might be beneficial for spinal cord repair, we have studied the effects of granulocyte‐macrophage colony stimulating factor (GM‐CSF) in the same paraplegia model and in cell cultures. There was a significant, though transient, improvement of locomotor recovery after a single delayed intraperitoneal injection of 2 µg GM‐CSF, which also increased significantly the expression of Cr3 and brain‐derived neurotrophic factor (BDNF) by macrophages at the lesion site. At longer survival delays, axonal regeneration was significantly enhanced in GM‐CSF‐treated rats. In vitro, BV2 microglial cells expressed higher levels of BDNF in the presence of GM‐CSF and neurons cocultured with microglial cells activated by GM‐CSF generated more neurites, an effect blocked by a BDNF antibody. These experiments suggest that GM‐CSF could be an interesting treatment option for spinal cord injury and that its beneficial effects might be mediated by BDNF.—Bouhy, D., Malgrange, B., Multon, S., Poirrier, A. L., Scholtes, F., Schoenen, J., Franzen, R. Delayed GM‐CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages. FASEB J. 20, E493–E502 (2006)

Migraine preventive drugs differentially affect cortical spreading depression in rat.

Cortical spreading depression (CSD) is the most likely cause of the migraine aura. Drugs with distinct pharmacological properties are effective in the preventive treatment of migraine. To test the hypothesis that their common denominator might be suppression of CSD we studied in rats the effect of three drugs used in migraine prevention: lamotrigine which is selectively effective on the aura but not on the headache, valproate and riboflavin which have a non-selective effect. Rats received for 4 weeks daily intraperitoneal injections of one of the three drugs. For valproate and riboflavin we used saline as control, for lamotrigine its vehicle dimethyl sulfoxide. After treatment, cortical spreading depressions were elicited for 2h by occipital KCl application. We measured CSD frequency, its propagation between a posterior (parieto-occipital) and an anterior (frontal) electrode, and number of Fos-immunoreactive nuclei in frontal cortex. Lamotrigine suppressed CSDs by 37% and 60% at posterior and anterior electrodes. Valproate had no effect on posterior CSDs, but reduced anterior ones by 32% and slowed propagation velocity. Riboflavin had no significant effect at neither recording site. Frontal Fos expression was decreased after lamotrigine and valproate, but not after riboflavin. Serum levels of administered drugs were within the range of those usually effective in patients. Our study shows that preventive anti-migraine drugs have differential effects on CSD. Lamotrigine has a marked suppressive effect which correlates with its rather selective action on the migraine aura. Valproate and riboflavin have no effect on the triggering of CSD, although they are effective in migraine without aura. Taken together, these results are compatible with a causal role of CSD in migraine with aura, but not in migraine without aura.

Effect of systemic nitroglycerin on CGRP and 5-HT afferents to rat caudal spinal trigeminal nucleus and its modulation by estrogen

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraine patients a delayed attack of unknown mechanism. After puberty migraine is more prevalent in women. Attacks can be triggered by abrupt falls in plasma estrogen levels, which accounts in part for sexual dimorphism, but lacks an established neurobiological explanation. We studied the effect of nitroglycerin on the innervated area of calcitonin gene‐related peptide (CGRP) and serotonin‐immunoreactive afferents to the superficial laminae of the spinal portion of trigeminal nucleus caudalis, and its modulation by estrogen. In male rats, nitroglycerin produced after 4 h a significant decrease of the area innervated by CGRP‐immunoreactive afferents and an increase of that covered by serotonin‐immunoreactive fibres. These effects were not observed in the superficial laminae of thoracic dorsal horns. The effect of nitroglycerin was similar in ovariectomized females. In estradiol‐treated ovariectomized females the area in the spinal portion of trigeminal nucleus caudalis laminae I–II covered by CGRP‐immunoreactive fibres was lower and that of serotonin‐immunoreactive fibres was higher than in males and for both transmitters not significantly changed after nitroglycerin. The bouton size of CGRP profiles was smaller in estradiol‐treated ovariectomized females, whereas after nitroglycerin it decreased significantly but only in males and ovariectomized females. Nitroglycerin, i.e. nitric oxide, is thus able to differentially influence afferent fibres in the superficial laminae of rat spinal trigeminal nucleus caudalis. Estradiol modulates the basal expression of these transmitters and blocks the nitroglycerin effect. These data may contribute to understanding the mechanisms by which estrogens influence migraine severity and the triggering of attacks by nitric oxide.

The effect of treadmill training on motor recovery after a partial spinal cord compression-injury in the adult rat.

Locomotor training on a treadmill is a therapeutic strategy used for several years in human paraplegics in whom it was shown to improve functional recovery mainly after incomplete spinal cord lesions. The precise mechanisms underlying its effects are not known. Experimental studies in adult animals were chiefly performed after complete spinal transections. The objective of this experiment was to assess the effects of early treadmill training on recovery of spontaneous walking capacity after a partial spinal cord lesion in adult rats. Following a compression-injury by a subdurally inflated microballoon, seven rats were trained daily on a treadmill with a body weight support system, whereas six other animals were used as controls and only handled. Spontaneous walking ability in an open field was compared weekly between both groups by two blinded observers, using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. Mean BBB score during 12 weeks was globally significantly greater in the treadmill-trained animals than in the control group, the benefit of training appearing as early as the 2nd week. At week 7, locomotor recovery reached a plateau in both animal groups, but remained superior in trained rats. Daily treadmill training started early after a partial spinal cord lesion in adult rats, which accelerates recovery of locomotion and produces a long-term benefit. These findings in an animal model mimicking the closed spinal cord injury occurring in most human paraplegics are useful for future studies of optimal locomotor training programs, their neurobiologic mechanisms, and their combination with other treatment strategies.

Vagus nerve stimulation in awake rats reduces formalin-induced nociceptive behaviour and fos-immunoreactivity in trigeminal nucleus caudalis

&NA; Besides its well‐established efficacy in epilepsy, vagus nerve stimulation (VNS) may be of potential interest in pain treatment. It has, however, not yet been assessed in animal pain models with the devices and stimulation protocols used in humans. We have therefore studied in awake rats the effects of left cervical VNS on trigeminal nociception using an implantable electrode and stimulator (NCP‐Cyberonics®). VNS was applied for 24 h at 2 mA intensity, 20 Hz frequency, 0.5 ms pulse width and a duty cycle of 20 s ON/18 s OFF. As a nociceptive stimulus, we injected formalin into the left mystacial vibrissae, assessed behaviour for 45 min and sacrificed the animals 45 min later. Fos‐immunoreactive (Fos‐Ir) neurons were counted in laminae I–II of trigeminal nucleus caudalis (TNC) on both sides. We used three groups of control animals: VNS without formalin, formalin without VNS and sham VNS (implanted without stimulation or formalin). Whereas sham VNS had no significant effect, VNS alone increased Fos expression in ipsilateral TNC in addition to the expected increase in nucleus tractus solitarius. It also significantly attenuated the increase of Fos‐Ir neurons observed in ipsilateral TNC laminae I–II after formalin injection. If the proper VNS effect on Fos‐expression was subtracted, the reduction of formalin‐induced nociceptor activation was 55%. VNS also reduced nociceptive behaviour on average by 96.1% during the early phase (0–6 min) and by 60.7% during the late phase (6–45 min) after the formalin injection. These results suggest that VNS applied with a device used in human therapy may have in awake rats a significant antinociceptive effect in a model of trigeminal pain.

Lack of estrogen increases pain in the trigeminal formalin model: a behavioural and immunocytochemical study of transgenic ArKO mice

&NA; In order to examine the effect of estrogen on facial pain, we first compared the face‐rubbing evoked by a formalin injection in the lip of aromatase‐knockout (ArKO) mice, lacking endogenous estrogen production, 17β‐estradiol‐treated ArKO mice (ArKO‐E2) and wild‐type (WT) littermates. During the ‘acute’ phase of pain the time spent rubbing was similar in the three groups, whereas during the following ‘interphase’ and the second phase of pain, grooming was increased in ArKO mice. Estradiol‐treatment restored a behaviour similar to WT group. To better understand estrogens modulation on pain processes, we examined changes in 5‐HT and CGRP innervations of trigeminal nucleus caudalis (TNC) in ArKO, ArKO‐E2 and WT groups sacrified during the interphase. Whereas serotonin and CGRP immunoreactivities were comparable in WT and ArKO non‐injected control groups, our data showed that 9 min after formalin injection, the density of serotoninergic terminals increased significantly in WT, but not in ArKO mice, while that of CGRP‐immunoreactive fibers was lower in WT than in ArKO mice on the injected side. Estradiol‐treatment only partially reversed these changes in ArKO‐E2 mice. We conclude that estrogen deprivation in ArKO mice can be responsible for increased nociceptive response and that it is accompanied by transmitter changes favouring pro‐ over anti‐nociceptive mechanisms in TNC during interphase of the formalin model. That estradiol‐treatment completely reverses the behavioural abnormality suggests that estrogens absence produces chiefly functional activation‐dependent changes. However, the fact that the immunohistochemical abnormalities were not totally normalized by estradiol‐treatment suggested that some permanent developmental alterations may occur in ArKO mice.

Effect of systemic kynurenine on cortical spreading depression and its modulation by sex hormones in rat

BACKGROUND The aura symptoms in migraine are most likely due to cortical spreading depression (CSD). CSD is favored by NMDA receptor activation and increased cortical excitability. The latter probably explains why migraine with aura may appear when estrogen levels are high, like during pregnancy. Kynurenic acid, a derivative of tryptophan metabolism, is an endogenous NMDA receptor antagonist whose cerebral concentrations can be augmented by systemic administration of its precursor L-kynurenine. OBJECTIVE To determine if exogenous administration of L-kynurenine is able to influence KCl-induced CSD in rat, if the effect is sex-dependent and if it differs in females between the phases of the estrous cycle. METHODS Adult Sprague-Dawley rats (n=8/group) received intraperitoneal (i.p.) injections of L-kynurenine (L-KYN, 300 mg/kg), L-KYN combined with probenecid (L-KYN+PROB) that increases cortical concentration of KYNA by blocking its excretion from the central nervous system, probenecid alone (PROB, 200 mg/kg) or NaCl. Cortical kynurenic acid concentrations were determined by HPLC (n=7). Thirty minutes after the injections, CSDs were elicited by application of 1M KCl over the occipital cortex and recorded by DC electrocorticogram. In NaCl and L-KYN groups, supplementary females were added and CSD frequency was analyzed respective to the phases of the estrous cycle determined by vaginal smears. RESULTS In both sexes, PROB, L-KYN and L-KYN+PROB increased cortical kynurenic acid level. PROB, L-KYN and L-KYN+PROB with increasing potency decreased CSD frequency in female rats, while in males such an effect was significant only for L-KYN+PROB. The inhibitory effect of L-KYN on CSD frequency in females was most potent in diestrus. CONCLUSION L-Kynurenine administration suppresses CSD, most likely by increasing kynurenic acid levels in the cortex. Females are more sensitive to this suppressive effect of L-kynurenine than males. These results emphasize the role of sex hormones in migraine and open interesting novel perspectives for its preventive treatment.

Repetitive transcranial magnetic stimulation improves open field locomotor recovery after low but not high thoracic spinal cord compression-injury in adult rats

Electromagnetic fields are able to promote axonal regeneration in vitro and in vivo. Repetitive transcranial magnetic stimulation (rTMS) is used routinely in neuropsychiatric conditions and as an atraumatic method to activate descending motor pathways. After spinal cord injury, these pathways are disconnected from the spinal locomotor generator, resulting in most of the functional deficit. We have applied daily 10 Hz rTMS for 8 weeks immediately after an incomplete high (T4–5; n = 5) or low (T10–11; n = 6) thoracic closed spinal cord compression‐injury in adult rats, using 6 high‐ and 6 low‐lesioned non‐stimulated animals as controls. Functional recovery of hindlimbs was assessed using the BBB locomotor rating scale. In the control group, the BBB score was significantly better from the 7th week post‐injury in animals lesioned at T4–5 compared to those lesioned at T10–11. rTMS significantly improved locomotor recovery in T10–11‐injured rats, but not in rats with a high thoracic injury. In rTMS‐treated rats, there was significant positive correlation between final BBB score and grey matter density of serotonergic fibres in the spinal segment just caudal to the lesion. We propose that low thoracic lesions produce a greater functional deficit because they interfere with the locomotor centre and that rTMS is beneficial in such lesions because it activates this central pattern generator, presumably via descending serotonin pathways. The benefits of rTMS shown here suggest strongly that this non‐invasive intervention strategy merits consideration for clinical trials in human paraplegics with low spinal cord lesions.

Influence of ovarian hormones on cortical spreading depression and its suppression by L-kynurenine in rat.

Migraine is sexually dimorphic and associated in 20–30% of patients with an aura most likely caused by cortical spreading depression (CSD). We have previously shown that systemic L-kynurenine (L-KYN), the precursor of kynurenic acid, suppresses CSD and that this effect depends on the stage of the estrous cycle in female rats. The objectives here are to determine the influence of ovarian hormones on KCl-induced CSD and its suppression after L-KYN by directly modulating estradiol or progesterone levels in ovariectomized rats. Adult female rats were ovariectomized and subcutaneously implanted with silastic capsules filled with progesterone or 17β-estradiol mixed with cholesterol, with cholesterol only or left empty. Two weeks after the ovariectomy/capsule implantation, the animals received an i.p. injection of L-KYN (300 mg/kg) or NaCl as control. Thirty minutes later CSDs were elicited by applying KCl over the occipital cortex and recorded by DC electrocorticogram for 1 hour. The results show that both estradiol and progesterone increase CSD frequency after ovariectomy. The suppressive effect of L-KYN on CSD frequency, previously reported in normal cycling females, is not found anymore after ovariectomy, but reappears after progesterone replacement therapy. Taken together, these results emphasize the complex role of sex hormones on cortical excitability. The CSD increase by estradiol and, more surprisingly, progesterone may explain why clinically migraine with aura appears or worsens during pregnancy or with combined hormonal treatments.