Plasma corticosterone levels in mouse models of pain

Background: Pain markedly activates the hypothalamic-pituitary-adrenal (HPA) axis and increases plasma corticosterone release interfering significantly with nociceptive behaviour as well as the mechanism of action of analgesic drugs. Aims/Methods: In the present study, we monitored the time course of circulating corticosterone in two mouse strains (C57Bl/6 and Balb/C) under different pain models. In addition, the stress response was investigated following animal handling, intrathecal (i.t.) manipulation and habituation to environmental conditions commonly used in nociceptive experimental assays. We also examined the influence of within-cage order of testing on plasma corticosterone. Results: Subcutaneous injection of capsaicin precipitated a prompt stress response whereas carrageenan and complete Freund's adjuvant induced an increased corticosterone release around the third hour post-injection. However, carrageenan induced a longer increased corticosterone in C57Bl/6 mice. In partial sciatic nerve ligation, neuropathic pain model corticosterone increased only in the first days whereas mechanical hypersensitivity remained much longer. Animal handling also represents an important stressor whereas the i.t. injection per se does not exacerbate the handling-induced stress response. Moreover, the order of testing animals from the same cage does not interfere with plasma corticosterone levels in the intrathecal procedure. Animal habituation to the testing apparatus also does not reduce the immediate corticosterone increase as compared with non-habituated mice. Conclusion: Our data indicate that HPA axis activation in acute and chronic pain models is time dependent and may be dissociated from evoked hyperalgesia. Therefore, HPA-axis activation represents an important variable to be considered when designing experimental assays of persistent pain as well as for interpretation of data.

Functional and morphometric differences between the early and delayed use of phototherapy in crushed median nerves of rats

This study evaluated the functional and quantitative differences between the early and delayed use of phototherapy in crushed median nerves. After a crush injury, low-level laser therapy (GaAs) was applied transcutaneously at the injury site, 3 min daily, with a frequency of five treatments per week for 2 weeks. In the early group, the first laser treatment started immediately after surgery, and in the delayed group, after 7 days. The grasping test was used for functional evaluation of the median nerve, before, 10, and 21 days after surgery, when the rats were killed. Three segments of the median nerve were analyzed histomorphometrically by light microscopy and computer analysis. The following features were observed: myelinated fiber and axon diameters, myelin sheath area, g-ratio, density and number of myelinated fibers, and area and number of capillaries. In the proximal segment (site of crush), the nerves of animals submitted to early and delayed treatment showed myelinated fiber diameter and myelin sheath area significantly larger compared to the untreated group. In the distal segment, the myelin sheath area was significantly smaller in the untreated animals compared to the delayed group. The untreated, early, and delayed groups presented a 50, 57, and 81% degree of functional recovery, respectively, at 21 days after injury, with a significant difference between the untreated and delayed groups. The results suggest that the nerves irradiated with low-power laser exhibit myelinated fibers of greater diameter and a better recovery of function.

Estimulação elétrica de alta voltagem em nervo ciático de ratos: estudo pelo IFC

OBJETIVO: Analisar a eficiência do tratamento com a estimulação elétrica de alta voltagem (EEAV) em três diferentes locais, aplicada precocemente na regeneração do nervo ciático submetido à lesão por esmagamento, e avaliada através do índice funcional do ciático (IFC), em ratos. MÉTODO: Após o esmagamento, 57 ratos foram submetidos à EEAV catódica nos parâmetros: frequência de 50Hz, 100V de tensão, 20 minutos diários, 5 dias por semana. Os ratos foram divididos aleatoriamente em: grupo controle; grupo gânglio; grupo gânglio + músculo; grupo músculo e; grupo simulado. O IFC foi avaliado semanalmente durante sete semanas, partindo do pré-operatório até a 6ª semana pós-operatória. RESULTADOS: Em comparação ao grupo controle, os resultados mostraram desempenho significativamente superior do grupo gânglio nas três primeiras semanas, e do grupo gânglio + músculo na 3ª semana, enquanto o grupo músculo teve desempenho significativamente negativo na 4ª e 6ª semanas. CONCLUSÃO: a EEAV aplicada precocemente, foi positiva no tratamento da região da medula e gânglio da raiz nervosa do ciático com o eletrodo dispersivo na região lombar contralateral ou no músculo gastrocnêmio. Porém, proporcionou efeitos negativos no tratamento com eletrodo ativo no músculo gastrocnêmio e dispersivo na coxa contralateral. Nível de evidência II, Estudo prospectivo comparativo.

Ação do laser terapêutico e do ultrassom na regeneração nervosa periférica

OBJETIVO: Avaliar a ação precoce do laser terapêutico e do ultrassom no processo de regeneração de uma lesão experimental em ratos. MÉTODO: Utilizou-se 24 ratos. Dezoito foram submetidos ao procedimento cirúrgico de lesão do nervo ciático por compressão, através de uma pinça hemostática acima da fossa poplítea. Os animais foram divididos em três grupos com seis animais em cada. Grupo controle normal. GI: controle lesado sem intervenção terapêutica. GII: intervenção terapêutica do laser ArGaAl. GIII: intervenção terapêutica do ultrassom Pulsado. Iniciamos as intervenções terapêuticas 24 horas após a lesão, com aplicações diárias, por um período de quatorze dias consecutivos. RESULTADOS: Ao avaliar a perimetria dos músculos da coxa direita obteve-se os seguintes valores médios de diminuição (mm), para cada grupo GI: 0,45; GII: 0,42; GIII: 0,40. Quanto ao tempo de deslocamento tanto o GII e GIII apresentaram diferença significativa, quando comparados ao GI. Na avaliação final do IFC o GII sobressaiu ao GIII. Quanto a cicatrização observou-se grande melhora no GII e GIII. CONCLUSÃO: Os resultados evidenciaram que a recuperação nervosa foi maior com a aplicação do laser. Nível de evidência II, Estudos terapêuticos - Investigação dos resultados do tratamento

Relationship between pain and anxiety in rats

Clinically, it is well known that neuropathic pain often induces comorbid symptoms such as anxiety. In turn, also anxiety has been associated with a heightened experience of pain. Although, the link between pain and anxiety is well recognized in humans, the neurobiological basis of this relationship remains unclear. Therefore, the aim of the current study was to investigate the influence of neuropathic pain on anxiety and vice versa in rats by assessing not only pain-related behaviour but also by discovering possible key substrates which are responsible for the interrelation of pain and anxiety.rnIn rats with a chronic constriction of the sciatic nerve (CCI model) anxiety-like behaviour was observed. Since anxiety behaviour could be completely abolished after the treatment of the pure analgesic drugs gabapentin and morphine, we concluded that anxiety was caused by the strong persistent pain. Furthermore, we found that the neuropeptides oxytocin and vasopressin were upregulated in the amygdala of CCI rats, and the intra-amygdala treatment of an oxytocin antagonist but not the vasopressin antagonist could reduce anxiety-like behaviour in these animals, while no effect on mechanical hypersensitivity was observed. These data indicate that oxytocin is implicated in the underlying neuronal processes of pain-induced anxiety and helps to elucidate the pathophysiological mechanisms of neuropathic pain. rnTo assess the influence of trait anxiety on pain sensation in rats, we determined mechanical hypersensitivity after sciatic nerve lesion (CCI) in animals selectively bred for high anxiety or low anxiety behaviour. The paw withdrawal thresholds were significantly decreased in high anxiety animals in comparison to low anxiety animals 2 and 3 weeks after surgery. In a second model state anxiety was induced by the sub-chronic injection of the anxiogenic drug pentylentetrazol in naive rats. Pain response to mechanical stimuli was increased after pharmacologically-induced anxiety. These results provided evidence for the influence of both trait and state anxiety on pain sensation. rnThe studies contribute to the elucidation of the relationship between pain and anxiety. We investigated that the neuropathic pain model displays sensory as well as emotional factors of peripheral neuropathy. Changes in expression levels of neuropeptides in the central nervous system due to neuropathic pain may contribute to the pathophysiology of neuropathic pain and its related symptoms in animals which might also be relevant for human scenarios. The results of the current study also confirm that anxiety plays an important role in the perception of pain. rnA better understanding of pain behaviour in animals might improve the preclinical profiling of analgesic drugs during development. The study highlights the potential use of the rat model as a new preclinical tool to further investigate the link between pain and anxiety by determining not only the sensory reflexes after painful stimuli but also the more complex pain-related behaviour such as anxiety.rn

Newly designed, self-coiling catheters for regional anesthesia--an imaging study

A major concern with the use of continuous peripheral nerve block is the difficulty encountered in placing the catheters close enough to the nerves to accomplish effective analgesia. The aim of this study was to investigate if a self-coiling catheter would remain close to the sciatic nerve once introduced through needles placed under ultrasound guidance and if contrast dye injected through the pigtail catheter made direct contact to the nerves.

Microneurography in rats: a minimally invasive method to record single C-fiber action potentials from peripheral nerves in vivo

Microneurography is a method suitable for recording intraneural single or multiunit action potentials in conscious subjects. Microneurography has rarely been applied to animal experiments, where more invasive methods, like the teased fiber recording technique, are widely used. We have tested the feasibility of microneurographic recordings from the peripheral nerves of rats. Tungsten microelectrodes were inserted into the sciatic nerve at mid-thigh level. Single or multiunit action potentials evoked by regular electrical stimulation were recorded, digitized and displayed as a raster plot of latencies. The method allows unambiguous recording and recognition of single C-fiber action potentials from an in vivo preparation, with minimal disruption of the nerve being recorded. Multiple C-fibers can be recorded simultaneously for several hours, and if the animal is allowed to recover, repeated recording sessions can be obtained from the same nerve at the same level over a period of weeks or months. Also, single C units can be functionally identified by their changes in latency to natural stimuli, and insensitive units can be recognized as 'silent' nociceptors or sympathetic efferents by their distinctive profiles of activity-dependent slowing during repetitive electrical stimulation, or by the effect on spontaneous efferent activity of a proximal anesthetic block. Moreover, information about the biophysical properties of C axons can be obtained from their latency recovery cycles. Finally, we show that this preparation is potentially suitable for the study of C-fiber behavior in models of neuropathies and nerve lesions, both under resting conditions and in response to drug administration.

Excessive erythrocytosis in adult mice overexpressing erythropoietin leads to hepatic, renal, neuronal, and muscular degeneration

To investigate the consequences of inborn excessive erythrocytosis, we made use of our transgenic mouse line (tg6) that constitutively overexpresses erythropoietin (Epo) in a hypoxia-independent manner, thereby reaching hematocrit levels of up to 0.89. We detected expression of human Epo in the brain and, to a lesser extent, in the lung but not in the heart, kidney, or liver of tg6 mice. Although no acute cardiovascular complications are observed, tg6 animals have a reduced lifespan. Decreased swim performance was observed in 5-mo-old tg6 mice. At about 7 mo, several tg6 animals developed spastic contractions of the hindlimbs followed by paralysis. Morphological analysis by light and electron microscopy showed degenerative processes in liver and kidney characterized by increased vascular permeability, chronic progressive inflammation, hemosiderin deposition, and general vasodilatation. Moreover, most of the animals showed severe nerve fiber degeneration of the sciatic nerve, decreased number of neuromuscular junctions, and degeneration of skeletal muscle fibers. Most probably, the developing demyelinating neuropathy resulted in muscular degeneration demonstrated in the extensor digitorum longus muscle. Taken together, chronically increased Epo levels inducing excessive erythrocytosis leads to multiple organ degeneration and reduced life expectancy. This model allows investigation of the impact of excessive erythrocytosis in individuals suffering from polycythemia vera, chronic mountain sickness, or in subjects tempted to abuse Epo by means of gene doping.

Interventional management of hypervascular osseous metastasis: role of embolotherapy before orthopedic tumor resection and bone stabilization

OBJECTIVE: The purpose of this study was to evaluate, in relation to intraoperative estimated blood loss (EBL), the effectiveness of preoperative transcatheter arterial embolization of hypervascular osseous metastatic lesions before orthopedic resection and stabilization. MATERIALS AND METHODS: Between June 1987 and November 2007, 22 patients underwent transcatheter arterial embolization of tumors of the long bone, hip, or vertebrae before resection and stabilization. Osseous metastatic lesions from renal cell carcinoma, malignant melanoma, leiomyosarcoma, and prostate cancer were embolized. All patients were treated with a coaxial catheter technique with polyvinyl alcohol (PVA) particles alone or a combination of PVA particles and coils. After embolization, each tumor was angiographically graded according to devascularization (grades 1-3) based on tumor blush after contrast injection into the main tumor-feeding arteries. RESULTS: In patients with complete devascularization (grade 1), mean EBL was calculated to be 1,119 mL, whereas in patients with partial embolization (grades 2 and 3) EBL was 1,788 mL and 2,500 mL. With respect to intraoperative EBL, no significant difference between devascularization grades was found (p > 0.05). Moderate correlation (r = 0.51, p = 0.019) was observed between intraoperative EBL and tumor size before embolization. Only low correlation (r = 0.44, p = 0.046) was found between intraoperative EBL and operating time. Major complications included transient palsy of the sciatic nerve and gluteal abscess in one patient. CONCLUSION: The results of this study support the concept that there is no statistically significant difference among amounts of intraoperative EBL with varying degrees of embolization.

Mechanical modeling of Intraneural Ganglion Cyst

Intraneural Ganglion Cyst is a 200 year old mystery related to nerve injury which is yet to be solved. Current treatments for the above problem are relatively simple procedures related to removal of cystic contents from the nerve. However, these treatments may result into neuropathic pain and recurrence of the cyst. The articular theory proposed by Spinner et al., (Spinner et al. 2003) takes into consideration the neurological deficit in Common Peroneal Nerve (CPN) branch of the sciatic nerve and affirms that in addition to the above treatments, ligation of articular branch results into foolproof eradication of the deficit. Mechanical Modeling of the Affected Nerve Cross Section will reinforce the articular theory (Spinner et al. 2003). As the cyst propagates, it compresses the neighboring fascicles and the nerve cross section appears like a signet ring. Hence, in order to mechanically model the affected nerve cross section; computational methods capable of modeling excessively large deformations are required. Traditional FEM produces distorted elements while modeling such deformations, resulting into inaccuracies and premature termination of the analysis. The methods described in this Master’s Thesis are effective enough to be able to simulate such deformations. The results obtained from the model adequately resemble the MRI image obtained at the same location and shows an appearance of a signet ring. This Master’s Thesis describes the neurological deficit in brief followed by detail explanation of the advanced computational methods used to simulate this problem. Finally, qualitative results show the resemblance of mechanical model to MRI images of the Nerve Cross Section at the same location validating the capability of these methods to study this neurological deficit.

Operative treatment of congenital hip osteoarthritis with high hip luxation (Crowe type IV)

OBJECTIVE The aim of the therapy is mechanical and functional stabilization of high dislocated hips with dysplasia coxarthrosis using total hip arthroplasty (THA). INDICATIONS Developmental dysplasia of the hip (DDH) in adults, symptomatic dysplasia coxarthrosis, high hip dislocation according to Crowe type III/IV, and symptomatic leg length inequality. CONTRAINDICATIONS Cerebrospinal dysfunction, muscular dystrophy, apparent disturbance of bone metabolism, acute or chronic infections, and immunocompromised patients. SURGICAL TECHNIQUE With the patient in a lateral decubitus position an incision is made between the anterior border of the gluteus maximus muscle and the posterior border of the gluteus medius muscle (Gibson interval). Identification of the sciatic nerve to protect the nerve from traction disorders by visual control. After performing trochanter flip osteotomy, preparation of the true actetabulum if possible. Implantation of the reinforcement ring, preparation of the femur and if necessary for mobilization, resection until the trochanter minor. Test repositioning under control of the sciatic nerve. Finally, refixation of the trochanteric crest. POSTOPERATIVE MANAGEMENT During hospital stay, intensive mobilization of the hip joint using a continuous passive motion machine with maximum flexion of 70°. No active abduction and passive adduction over the body midline. Maximum weight bearing 10-15 kg for 8 weeks, subsequently, first clinical and radiographic follow-up and deep venous thrombosis prophylaxis until full weight bearing. RESULTS From 1995 to 2012, 28 THAs of a Crow type IV high hip-dislocation were performed in our institute. Until now 14 patients have been analyzed during a follow-up of 8 years in 2012. Mid-term results showed an improvement of the postoperative clinical score (Merle d'Aubigné score) in 86 % of patients. Good to excellent results were obtained in 79 % of cases. Long-term results are not yet available. In one case an iatrogenic neuropraxia of the sciatic nerve was observed and after trauma a redislocation of the arthroplasty appeared in another case. In 2 cases an infection of the THA appeared 8 and 15 months after index surgery. No pseudoarthrosis of the trochanter or aseptic loosening was noticed.

THE EXPRESSION PROFILE OF A GWAS DETECTED GENE (NINJ2) IN THE BRAIN OF A MOUSE MODEL OF ISCHEMIC STROKE

Stroke is the third leading cause of death and a major debilitating disease in the United States. Multiple factors, including genetic factors, contribute to the development of the disease. Genome-wide association studies (GWAS) have contributed to the identification of genetic loci influencing risk for complex diseases, such as stroke. In 2010, a GWAS of incident stroke was performed in four large prospective cohorts from the USA and Europe and identified an association of two Single Nucleotide Polymorphisms (SNPs) on chromosome 12p13 with a greater risk of ischemic stroke in individuals of European and African-American ancestry. These SNPs are located 11 Kb upstream of the nerve injury-induced gene 2, Ninjurin2 (NINJ2), suggesting that this gene may be involved in stroke pathogenesis. NINJ2 is a cell adhesion molecule induced in the distal glial cells from a sciatic-nerve injury at 7-days after injury. In an effort to ascribe a possible role of NINJ2 in stroke, we have assessed changes in the level of gene and protein expression of NINJ2 following a time-course from a transiently induced middle cerebral artery ischemic stroke in mice brains. We report an increase in the gene expression of NINJ2 in the ischemic and peri-infarct (ipsilateral) cortical tissues at 7 and 14-days after stroke. We also report an increase in the protein expression of NINJ2 in the cortex of both the ipsilateral and contralateral cortical tissues at the same time-points. We conclude that the expression of NINJ2 is regulated by an ischemic stroke in the cortex and is consistent with NINJ2 being involved in the recovery time-points of stroke.

Neuromuscular disuse and atrophy: {\it In vivo\/} magnetic resonance studies of intramuscular lipid metabolism and fluid shift changes

We postulated that neuromuscular disuse results in deleteriously affected tissue-vascular fluid exchange processes and subsequently damages the important oxidative bioenergetic process of intramuscular lipid metabolism. The in-depth research reported in the literature is somewhat limited by the ex vivo nature and sporadic time-course characterization of disuse atrophy and recovery. Thus, an in vivo controlled, localized animal model of disuse atrophy was developed in one of the hindlimbs of laboratory rabbits (employing surgically implanted tetrodotoxin (TTX)-filled mini-osmotic pump-sciatic nerve superfusion system) and tested repeatedly with magnetic resonance (MR) throughout the 2-week period of temporarily induced disuse and during the recovery period (following explantation of the TTX-filled pump) for a period of 3 weeks. Controls consisted of saline/"sham"-implanted rabbit hindlimbs. The validity of this model was established with repeated electrophysiologic nerve conduction testing using a clinically appropriate protocol and percutaneously inserted small needle stimulating and recording electrodes. Evoked responses recorded from proximal (P) and distal (D) sites to the sciatic nerve cuff in the TTX-implanted group revealed significantly decreased (p $<$ 0.001) proximal-to-distal (P/D) amplitude ratios (as much as 50-70% below Baseline/pre-implanted and sham-implanted group values) and significantly increased (p $<$ 0.01) differential latency (PL-DL) values (as much as 1.5 times the pre- and sham-implanted groups). By Day 21 of recovery, observed P/D and PL-DL levels matched Baseline/sham-implemented levels. MRI-determined cross-sectional area (CSA) values of Baseline/pre-implanted, sham- or TTX-implanted, and recovering/explanted and the corresponding contralateral hindlimb tibialis anterior (TA) muscles normalized to tibial bone (TB) CSA (in TA/TB ratios) revealed that there was a significant decline (indicative of atrophic response) from pre- and sham-implanted controls by as much as 20% (p $<$ 0.01) at Day 7 and 50-55% (p $<$ 0.001) at Day 13 of TTX-implantation. In the non-implanted contralaterals, a significant increase (indicative of hypertrophic response) by as much as 10% (p $<$ 0.025) at Day 7 and 27% (p $<$ 0.001) at Day 13 + TTX was found. The induced atrophic/hypertrophic TA muscles were observed to be fully recovered by Day 21 post-explantation as evidenced by image TA/TB ratios. End-point biopsy results from a small group of rabbits revealed comprehensive atrophy of both Type I and Type II fibers, although the heterogeneity of the response supports the use of image-guided, volume-localized proton magnetic resonance spectroscopy (MRS) to noninvasively assess tissue-level metabolic changes. MRS-determined results of a 0.25cc volume of tissue within implanted limb TA muscles under resting/pre-ischemic, ischemic-stressed, and post-ischemic conditions at timepoints during and following disuse atrophy/recovery revealed significantly increased intramuscular spectral lipid levels, as much as 2-3 times (p $<$ 0.01) the Baseline/pre-implanted values at Day 7 and 6-7 times (p $<$ 0.001) at Day 13 + TTX, which approached normal levels (compared to pre- and sham-implanted groups) by Day 21 of post-explanation recovery. (Abstract shortened by UMI.) ^

Dysfunction of Cortical Dendritic Integration in Neuropathic Pain Reversed by Serotoninergic Neuromodulation

Neuropathic pain is caused by long-term modifications of neuronal function in the peripheral nervous system, the spinal cord, and supraspinal areas. Although functional changes in the forebrain are thought to contribute to the development of persistent pain, their significance and precise subcellular nature remain unexplored. Using somatic and dendritic whole-cell patch-clamp recordings from neurons in the anterior cingulate cortex, we discovered that sciatic nerve injury caused an activity-dependent dysfunction of hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the dendrites of layer 5 pyramidal neurons resulting in enhanced integration of excitatory postsynaptic inputs and increased neuronal firing. Specific activation of the serotonin receptor type 7 (5-HT7R) alleviated the lesion-induced pathology by increasing HCN channel function, restoring normal dendritic integration, and reducing mechanical pain hypersensitivity in nerve-injured animals in vivo. Thus, serotoninergic neuromodulation at the forebrain level can reverse the dendritic dysfunction induced by neuropathic pain and may represent a potential therapeutical target.

Stimulation of neurite outgrowth using an electrically conducting polymer

Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer—oxidized polypyrrole (PP)—has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(l-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 μm (n = 5643) compared with 9.5 μm (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid).