Jan H. Brakkee

Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function: Beneficial effects of melanocortins

A procedure for placing a crush lesion in the sciatic and tibial nerve of the rat based on anatomical landmarks is described. These crush lesions are used to study the process of regeneration of peripheral nervous tissue and the beneficial effects of melanocortins on speed and quality of nerve regeneration. A new precise and rapid method for testing the return of sensory function by a locally applied electric stimulus is discussed.

Exercise training improves functional recovery and motor nerve conduction velocity after sciatic nerve crush lesion in the rat

OBJECTIVE To observe the effects of exercise training on recuperation of sensorimotor function in the early phase of regeneration, and to monitor the long-term effects of exercise on electrophysiological aspects of the regenerating nerve. DESIGN After sciatic nerve crush in 20 male Wistar rats, one random selected group was subjected to 24 days of exercise training, whereas the other group served as sedentary controls. INTERVENTIONS Exercise training was induced for 24 days, starting the first postoperation day, by placing bottles of water at such a height that the exercising rats had to maximally erect on both hindpaws to drink. MAIN OUTCOME MEASURES Recovery of motor and sensory function in the early phase was monitored by analysis of the free walking pattern and the foot reflex withdrawal test, respectively. Electrophysiological measurements on postoperation days 50, 75, 100, 125, and 150 were used to evaluate the late phase of recovery of nerve conduction velocity. RESULTS During the early phase of the recovery period, exercise training enhanced functional recovery. The motor nerve conduction velocity (MNCV), as measured in the late phase of recovery, was significantly better in the trained group than in the control group (p < .01). CONCLUSIONS We conclude that exercise training enhances the return of sensomotoric function in the early phase of recovery from peripheral nerve lesion. Furthermore, these results suggest that the beneficial effects of 24 days of exercise training after crush persist in the late phase of peripheral nerve recovery.

Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome

Peripheral nerve regeneration has been studied extensively in the sciatic nerve crush model, at the level of both function and gene expression. The crush injury allows full recovery of sensory and motor function in about 3 weeks as assessed by the foot reflex withdrawal test and De Medinacelli walking patterns. We used the recently developed CatWalk paradigm to study walking patterns in more detail in mice and rats. We found that, following the recovery of sensory function, the animals developed a state of mechanical allodynia, which retreated slowly over time. The motor function, although fully recovered with the conventional methods, was revealed to be still impaired because the animals did not put weight on their previously injured paw. The development of neuropathic pain following successful sensory recovery has not been described before in crush-lesioned animals and may provide an important new parameter to assess full sensory recovery.

Characterization of melanocortin receptor ligands on cloned brain melanocortin receptors and on grooming behavior in the rat

Since the melanocortin MC3 and melanocortin MC4 receptors are the main melanocortin receptor subtypes expressed in rat brain, we characterized the activity and affinity of nine melanocortin receptor ligands using these receptors in vitro, as well as their activity in a well-defined melanocortin-induced behavior in the rat: grooming behavior. We report here that [D-Tyr4]melanotan-II and RMI-2001 (Ac-cyclo-[Cys4, Gly5, D-Phe7, Cys10]alpha-MSH-NH2) have significantly higher affinity and potency on the rat melanocortin MC4 receptor as compared to the rat melanocortin MC3 receptor. Nle-gamma-MSH (melanocyte-stimulating hormone) was the only ligand with higher affinity and potency on the rat melanocortin MC3 receptor. The potency order of melanocortin MC4 receptor agonists, but not that of melanocortin MC3 receptor agonists, fitted with the potency of these ligands to stimulate grooming behavior, when administered intracerebroventricularly. SHU9119 (Ac-cyclo-[Nle4, Asp5, D-Nal(2)7, Lys10]alpha-MSH-(4-10)-NH2) and RMI-2005 (Ac-cyclo-[Cys4, Gly5, D-Na](2)7, Nal(2)9, Cys10]alpha-MSH-(4-10)-NH2) were able to inhibit alpha-MSH-induced melanocortin receptor activity in vitro, as well as alpha-MSH-induced grooming behavior. Melanotan-II, [Nle4-D-Phe7]alpha-MSH and RMI-2001 were also effective in inducing grooming behavior when administered intravenously. In the absence of purely selective melanocortin MC(3/4) receptor ligands, we demonstrated that careful comparison of ligand potencies in vitro with ligand potencies in vivo, could identify which melanocortin receptor subtype mediated alpha-MSH-induced grooming behavior. Furthermore, blockade of novelty-induced grooming behavior by SHU9119 demonstrated that this physiological stress response is mediated via activation of the melanocortin system.

Identification of antagonists for melanocortin MC3, MC4 and MC5 receptors

Antagonists for the melanocortin receptor family were identified by analysis of the effects of four melanocortin analogues on alpha-MSH(alpha-melanocyte-stimulating hormone)-induced cAMP accumulation in 293 human embryonal kidney (HEK) cells that expressed either the rat melanocortin MC3 receptor, the human melanocortin MC4 receptor or the ovine melanocortin MC5 receptor. Two peptides, [D-Arg8]ACTH(adrenocorticotrope hormone)-(4-10) and [Pro8,10,Gly9]ACTH-(4-10), antagonized the action of alpha-MSH on the melanocortin MC4 and MC5 receptors, but not the melanocortin MC3 receptor. [Ala6]ACTH-(4-10) inhibited the alpha-MSH activation of the melanocortin MC3 and MC5, but only weakly antagonized the activation of the melanocortin MC4 receptor. [Phe-I7]ACTH-(4-10) antagonized the melanocortin MC3, MC4 and MC5 receptors equally well. These antagonists were also tested to block a behavioral response induced by alpha-MSH. alpha-MSH-induced excessive grooming behavior in rats was inhibited by [Phe-I7]ACTH-(4-10), [D-Arg8]ACTH-(4-10) and [Pro8,10,Gly9]ACTH-(4-10), but not by [Ala6]ACTH-(4-10). This suggests that alpha-MSH-induced excessive grooming behavior is mediated by melanocortin MC4 receptors.

Expression of melanocortin receptors and pro-opiomelanocortin in the rat spinal cord in relation to neurotrophic effects of melanocortins

Although neurotrophic effects of alpha-melanocyte-stimulating hormone (alpha-MSH) are well established, the mechanism underlying these effects is unknown. To identify candidate components of the signaling system that may mediate these effects, in the present study rat spinal cord, dorsal root ganglia, sciatic nerve and soleus muscle were analysed for the expression of the neural MC3, MC4 and MC5 receptors and for the expression of the melanocortin precursor pro-opiomelanocortin (POMC). In rat lumbar spinal cord, the MC4 receptor was the only MC receptor subtype for which mRNA was detectable using RNAse protection assays. In situ binding studies using 125I-NDP-MSH, a synthetic alpha-MSH analogue, demonstrated MC receptor protein in the rat spinal cord, predominantly localised in substantia gelatinosa and area X, surrounding the central canal. Furthermore, POMC mRNA was demonstrated in rat spinal cord and dorsal root ganglia. These findings suggest a functional melanocortin system in the rat spinal cord, that might be involved in peripheral nerve repair. Regulation of POMC or MC receptor transcripts does not appear to be involved in the response to peripheral nerve crush in rats, since no change in mRNA expression patterns was detected after sciatic nerve crush, using quantitative RNAse protection assays. Nevertheless, subtle changes in melanocortin receptor binding did occur postsurgically in several regions of the spinal cord in both sham-operated and sciatic nerve-lesioned rats. The robust expression of MC receptor protein in spinal cord regions that are generally associated with nociception suggests a potentially broader involvement of endogenous melanocortins in spinal pathways which mediate the responses to peripheral injury, in addition to any direct melanocortin effects on sprouting and neurite outgrowth.

Melanocortin Receptor 4 Deficiency Affects Body Weight Regulation, Grooming Behavior, and Substrate Preference in the Rat

Obesity is caused by an imbalance between energy intake and expenditure and has become a major health‐care problem in western society. The central melanocortin system plays a crucial role in the regulation of feeding and energy expenditure, and functional loss of melanocortin receptor 4 (MC4R) is the most common genetic cause of human obesity. In this study, we present the first functional Mc4r knockout model in the rat, resulting from an N‐ethyl‐N‐nitrosourea mutagenesis–induced point mutation. In vitro observations revealed impaired membrane‐binding and subsequent nonfunctionality of the receptor, whereas in vivo observations showed that functional loss of MC4R increased body weight, food intake, white adipose mass, and changed substrate preference. In addition, intracerebroventricular (ICV) administration of Agouti‐Related Protein79–129 (AgRP79–129), an MC4R inverse agonist, or Melanotan‐II (MTII), an MC4R agonist, did affect feeding behavior in wild‐type rats but not in homozygous mutant rats, confirming complete loss of MC4R function in vivo. Finally, ICV administration of MTII induced excessive grooming behavior in wild‐type rats, whereas this effect was absent in homozygous mutant rats, indicating that MTII‐induced grooming behavior is exclusively regulated via MC4R pathways. Taken together, we expect that the MC4R rat model described here will be a valuable tool for studying monogenic obesity in humans. More specifically, the relative big size and increased cognitive capacity of rats as compared to mice will facilitate complex behavioral studies and detailed mechanistic studies regarding central function of MC4R, both of which ultimately may help to further understand the specific mechanisms that induce obesity during loss of MC4R function.

Putative neurotrophic factors and functional recovery from peripheral nerve damage in the rat

1 In rats, recovery of sensory‐motor function following a crush lesion of the sciatic or tibial nerve was monitored by measuring foot reflex withdrawal from a local noxious stimulation of the foot sole. 2 Putative neurotrophic compounds were tested on this functional recovery model: melanocortins (peptides derived from ACTH (corticotropin) and α‐MSH (melanotropin)), gangliosides and nimodipine were effective whereas isaxonine and TRH (thyrotropin releasing hormone) were not. 3 Structure‐activity studies with melanocortins revealed a similar effectiveness of α‐MSH, [N‐Leu4, d‐Phe7]‐α‐MSH, desacetyl‐α‐MSH and the ACTH4–9 analogue ORG 2766, questioning the validity of the previously suggested notion that the melanotrophic properties of these peptides are responsible for their neurotrophic effect. 4 As recovery of function after peripheral nerve damage follows a similar time course in hypophysectomized (five days post operation) and sham‐operated rats, effective melanocortin therapy does not mimic an endogenous peptide signal in the repair process from pituitary origin. 5 Subcutaneous treatment with ORG 2766 (7.5 μg kg−1 48 h−1) facilitates recovery of function following peripheral nerve damage in young (6–7 weeks old), mature (5 month old) and old (20 month old) rats. 6 In view of the diversity in structure of the effective neurotrophic factors and the complexity of nerve repair, the present data support the notion that peripheral nerve repair may be facilitated by different humoral factors likely to be active on different aspects of the recovery process.

α-MSH and Org.2766 in peripheral nerve regeneration: different routes of delivery

The efficacy of melanocortins (alpha-MSH and an ACTH-(4-9) analog, Org.2766) in accelerating functional recovery from sciatic nerve damage following various types of subcutaneous and oral administration was assessed in the rat. Furthermore, the effectiveness of the local delivery of melanocortins to the site of injury was examined. An accelerated recovery was evident following subcutaneous constant delivery of Org.2766 from an osmotic mini-pump and from biodegradable polymere microspheres, and was as effective as repeated subcutaneous injections of alpha-MSH or Org.2766. Oral administration of Org.2766 was ineffective. Local application of Org.2766, achieved by wrapping a peptide-impregnated biodegradable gelatine foam matrix around the site of injury, facilitated recovery as well. The biodegradable microspheres and gelatine foam matrix may be of importance in eventual clinical use as effective vehicles for administration of melanocortins in the treatment of peripheral nerve damage.

Characterization of febrile seizures and febrile seizure susceptibility in mouse inbred strains

Febrile seizures (FS) are the most prevalent seizures in children. Although FS are largely benign, complex FS increase the risk to develop temporal lobe epilepsy (TLE). Studies in rat models for FS have provided information about functional changes in the hippocampus after complex FS. However, our knowledge about the genes and pathways involved in the causes and consequences of FS is still limited. To enable molecular, genetic and knockout studies, we developed and characterized an FS model in mice and used it as a phenotypic screen to analyze FS susceptibility. Hyperthermia was induced by warm air in 10‐ to 14‐day‐old mice and induced FS in all animals. Under the conditions used, seizure‐induced behavior in mice and rats was similar. In adulthood, treated mice showed increased hippocampal Ih current and seizure susceptibility, characteristics also seen after FS in rats. Of the seven genetically diverse mouse strains screened for FS susceptibility, C57BL/6J mice were among the most susceptible, whereas A/J mice were among the most resistant. Strains genetically similar to C57BL/6J also showed a susceptible phenotype. Our phenotypic data suggest that complex genetics underlie FS susceptibility and show that the C57BL/6J strain is highly susceptible to FS. As this strain has been described as resistant to convulsants, our data indicate that susceptibility genes for FS and convulsants are distinct. Insight into the mechanisms underlying seizure susceptibility and FS may help to identify markers for the early diagnosis of children at risk for complex FS and TLE and may provide new leads for treatment.