Axonal regeneration through the fibrous scar in lesioned goldfish spinal cord.

Spontaneous nerve regeneration beyond the scar frequently occurs in fish after spinal cord lesions, in contrast to mammals. Here we examined the spatiotemporal relationship between the fibrous scar and axonal regeneration in the goldfish. Within 1 week after hemisection of the spinal cord, the open wound was closed by a fibrous scar that was demarcated from the surrounding nervous tissue by the glia limitans, which was immunoreactive for laminin. Within 1 week after hemisection, regenerating axons entered the fibrous scar, and were surrounded by laminin-coated tubular structures continuous with the glia limitans. Regenerating axons that initially entered the fibrous scar were usually accompanied by glial processes. Within 2-3 weeks after hemisection, the tubular structures became enlarged, and the regenerating axons increased in number, fasciculating in the tubules. Glial processes immunoreactive for glial fibrillary acid protein and 5-hydroxytryptamine neurons then entered the tubular structures to associate with the regenerating axons. The tubular structures developed further, creating tunnels that penetrated the fibrous scar, through which the regenerating axons passed. At 6-12 weeks after hemisection, the fibrous scar was smaller and the enlarged tunnels contained many glial processes and several axons. The findings of present study demonstrated that, following spinal lesions in goldfish, regenerating axons enter and pass the scar tissue. The regenerating axons first enter the fibrous scar with glial elements and then grow through laminin-coated tubular structures within the fibrous scar. Invasion by glial processes and neuronal elements into the tubular structures reduces the fibrous scar area and allows for more regenerating axons to pass beyond the fibrous scar.

Impaired oxygen utilization in skeletal muscle of CRPS I patients.

BACKGROUND: The purpose of this study was to evaluate oxygen extraction and utilization in end stage chronic complex regional pain syndrome type I (CRPS I) patients undergoing amputation and to relate these to muscle histology of the amputated limb. MATERIALS AND METHODS: In 25 patients with severe CRPS I requiring amputation of the affected limb venous blood samples and in 11 patients skeletal muscle specimens were analyzed. RESULTS: The mean venous oxygen saturation (S(v)O(2)) value (94.3% ± 4.0%) of the affected limb was significantly higher than S(v)O(2) values found in healthy subjects (77.5% ± 9.8%) pointing to a severely decreased oxygen diffusion or utilization within the affected limb. Histologic analysis showed a significant decrease of type I fibers and a significant increase of type IIB fibers. Ultrastructural investigations of soleus skeletal muscle capillaries revealed thickened endothelial cells and thickened basement membranes. Muscle capillary densities were decreased in comparison with literature data. High venous oxygen saturation levels were partially explained by impaired diffusion of oxygen due to thickened basement membrane and decreased capillary density. CONCLUSION: This study shows that venous oxygen saturation is significantly increased in chronic end stage CRPS I patients corresponding with impaired oxygen diffusion. The abnormal skeletal muscle findings points to severe disuse but only partially explain the impaired diffusion of oxygen; mitochondrial dysfunction seems a likely explanation in addition.

Increased migration of IgA lymphocytes to VIP nerve fibers after DSS-induced colitis.

Immunoglobulin-positive lymphocytes are present close to vasoactive intestinal polypeptide-positive (VIP(+)) nerve fibers in the lamina propria of the intestinal tract, and have an important role in mucosal defense. The number of immunoglobulin A-positive (IgA(+)) cells close to the epithelial basement membrane and nerve fibers is increased by the administration of lipopolysaccharides, which induce IgA secretion into the intestinal lumen. The relationship between immunoglobulin-positive lymphocytes and the VIP(+) nerve fibers during inflammation, such as in inflammatory bowel disease, however, is not well known. The morphological relationship between immunoglobulin-positive cells and the basement membrane or the VIP(+) nerve fibers in the colon was examined using double immunofluorescent labeling in an inflammatory bowel disease mouse model created by oral administration of dextran sodium sulfate (DSS). DSS administration induced goblet cell loss, crypt loss, intestinal epithelium deformation and infiltration of inflammatory cells in the mucosa. In the colon, the number and percentage of IgA(+) lymphocytes close to the basement membrane and the VIP(+) nerve fibers in the lamina propria increased after DSS administration, in parallel with the pathologic progress in the inflamed tissue. On the other hand, the percentage of immunoglobulin G-positive (IgG(+)) lymphocytes close to the basement membrane and the VIP(+) nerve fibers decreased, although the total number of IgG(+) lymphocytes in the lamina propria increased. We suggest that the immunoglobulin-producing lymphocytes and enteric nerve fibers in the colon normally have a close morphological relationship, and that this relationship is reinforced in a cell-specific manner during inflammation.

Mitochondrial dysfunction in muscle tissue of complex regional pain syndrome type I patients.

Reactive oxygen species (ROS) are known to be involved in the pathophysiology of complex regional pain syndrome type I (CRPS I). Since the mitochondrial respiratory chain is a major source of ROS, we hypothesized that mitochondria play a role in the pathophysiology of CRPS I. The hypothesis was tested by studying mitochondrial energy metabolism in muscle tissue from amputated limbs of CRPS I patients. We observed that mitochondria obtained from CRPS I muscle tissue displayed reduced mitochondrial ATP production and substrate oxidation rates in comparison to control muscle tissue. Moreover, we observed reactive oxygen species evoked damage to mitochondrial proteins and reduced MnSOD levels. It remains to be established if the mitochondrial dysfunction that is apparent at the end-stage of CRPS I is also present in earlier stages of the disease, or are secondary to CRPS I. The observation of a reduced mitochondrial energy production combined with reactive oxygen species induced damage in muscle tissue from CRPS I patients warrants further studies into the involvement of mitochondrial dysfunctioning in the pathophysiology of CRPS I.

Intra-arterial tert-Butyl-hydroperoxide infusion induces an exacerbated sensory response in the rat hind limb and is associated with an impaired tissue oxygen uptake.

The objective of this study was to investigate oxidative stress and oxygen extraction mechanisms in an animal model of continuous intra-arterial infusion of a free radical donor and in an in vitro model using isolated mitochondria. tert-Butyl-hydroperoxide (tert-BuOOH, 25 mM) was infused for 24 h in the left hind limb of rats to induce soft tissue damage (n = 8). After 7 days, we assessed local sensory response, tissue oxygen consumption, oxygen radicals, and antioxidant levels. In vitro mitochondrial function was measured after stimulation of isolated mitochondria of skeletal muscle cells with increasing doses of tert-BuOOH. tert-BuOOH infusion resulted in an increased skin temperature (p = 0.04), impaired function, and a significantly increased pain sensation (p = 0.03). Venous oxygen saturation levels (p = 0.01) and the antioxidant ceruloplasmin (p = 0.04) were increased. tert-BuOOH inhibited mitochondrial function in vitro. Induction of free radical formation in the rat hind limb results in an exacerbated sensory response and is associated with impaired oxygen extraction, which likely results from mitochondrial dysfunction caused by free radicals.

The oxidative response in the chronic constriction injury model of neuropathic pain.

BACKGROUND: In the chronic constriction injury model of rat neuropathic pain, oxidative stress as well as antioxidants superoxide dismutase and reduced glutathione (GSH) are important determinants of neuropathological and behavioral consequences. Studies of the chronic constriction injury model observed (indirect) signs of inflammation. We, therefore, investigated the level of oxidative stress and antioxidant enzymes in skeletal muscle tissue of the rat hind paw and (jugular vein) plasma at d 7 after nerve injury. MATERIALS AND METHODS: The level of reactive oxygen and nitrogen species (RONS) was determined as a measure of oxidative stress. Reduced GSH levels and the ceruloplasmin/transferrin ratio were determined as measures of overall antioxidant activity. RONS and overall antioxidant activity were measured in skeletal muscle tissue of the hind paw and jugular vein plasma. The level of RONS in muscle was determined using spin trapping combined with electron paramagnetic resonance spectroscopy. Using electron paramagnetic resonance spectroscopy, we also determined plasma levels of transferrin and ceruloplasmin. GSH levels were determined using high-performance liquid chromatography. RESULTS: In skeletal muscle tissue, the level of RONS was lower in nerve-injured hind paws than in controls. The plasma level (jugular vein) of RONS did not differ between nerve-injured and control rats. In skeletal muscle tissue, the level of GSH was higher in nerve-injured hind paws than in controls. The ceruloplasmin/transferrin ratio tended to be higher in (jugular vein) plasma of nerve-injured rats as compared to controls. CONCLUSIONS: This study shows that, at d 7 after nerve injury, oxidative stress-induced changes are present also in skeletal muscle tissue of the rat hind paw. Our findings of a decreased level of RONS in combination with an increased level of the antioxidant GSH suggest that an overshoot of antioxidant activity overrules initial oxidative stress.

Complex regional pain syndrome type I in children.

BACKGROUND: Complex Regional Pain Syndrome type I (CRPS I) is a potentially incapacitating syndrome which can occur after a minor injury or operation to a limb. It is a disorder characterized by pain, sensory and motor disturbances. CRPS I is well known in adults, but a relatively new diagnostic entity in children. The clinical presentation of CRPS I in children is, to some extent, different from adults and therefore sometimes not recognized early. The aim of this study was to search for differences in patient characteristics between children and adults with CRPS I. METHODS: We have performed a retrospective chart review of 78 children (age

Adult neurogenesis with 5-HT expression in lesioned goldfish spinal cord.

In contrast to mammals, spontaneous nerve regeneration occurs in the teleost spinal cord. In the present study, we examined whether neurogenesis is involved in posttraumatic regeneration in the goldfish spinal cord. In intact fish, many spinal cells positive for both a monoclonal neuronal marker (Hu) and bromodeoxyuridine (BrdU) were observed 24 h after i.p. injection of BrdU, suggesting that constant neurogenesis occurs in the goldfish spinal cord. After hemisection of the spinal cord, the number of spinal cells positive for Hu and BrdU was significantly increased around the lesion site. The number of Hu- and BrdU-positive cells reached the maximum level 7 days after hemisection. In intact fish, spinal cells positive for both Hu and BrdU were also observed 5 weeks after BrdU injection, suggesting that newborn neurons survive for a long time. Six weeks after hemisection, the number of surviving Hu- and BrdU-positive cells at the lesion site was significantly increased as compared with that in intact fish, and some of them were also positive for 5-HT. A retrograde tract tracing study showed that the 5-HT+ neurons were close to the regenerated axons passing through the lesion site. These results suggest that adult neurogenesis occurs in the goldfish spinal cord, and that neurogenesis is activated by spinal cord lesion. The newly produced neurons survive a long time at the lesion site, and might participate in the repair of injured tissue and in the regeneration of descending long axons beyond the lesion site.

Delayed recovery and late development of complex regional pain syndrome in patients with an isolated fracture of the distal radius: prediction of a regional inflammatory response by early signs.

We studied prospectively the regional inflammatory response to a unilateral distal radial fracture in 114 patients at eight to nine weeks after injury and again at one year. Our aim was to identify patients at risk for a delayed recovery and particularly those likely to develop complex regional pain syndrome. In order to quantify clinically the inflammatory response, a regional inflammatory score was developed. In addition, blood samples were collected from the antecubital veins of both arms for comparative biochemical and blood-gas analysis. The severity of the inflammatory response was related to the type of treatment (Kruskal-Wallis test, p = 0.002). A highly significantly-positive correlation was found between the regional inflammatory score and the length of time to full recovery (r(2) = 0.92, p = 0.01, linear regession). A regional inflammatory score of 5 points with a sensitivity of 100% but a specificity of only 16% also identified patients at risk of complex regional pain syndrome. None of the biochemical parameters studied correlated with regional inflammatory score or predicted the development of complex regional pain syndrome. Our study suggests that patients with a distal radial fracture and a regional inflammatory score of 5 points or more at eight to nine weeks after injury should be considered for specific anti-inflammatory treatment.

Subareolar breast abscesses: characteristics and results of surgical treatment.

Our objective was to describe the characteristics of subareolar breast abscesses and to analyze the results of surgical treatment in relation to the prevention of recurrences. Almost 70% of patients smoked more than 10 cigarettes a day. The recurrence rate after excision of the lactiferous ducts was 28% and after management without excision of the lactiferous ducts was 79% (p < 0.001). Gram-positive bacteria were isolated more frequently in primary subareolar breast abscesses (not significant). Anaerobic microorganisms were more frequently cultured in recurring subareolar breast abscesses (p = 0.02). Definitive treatment of subareolar breast abscesses should consist of excision of the affected lactiferous ducts.

Zymosan-induced generalized inflammation: experimental studies into mechanisms leading to multiple organ dysfunction syndrome.

Patients suffering from multiple organ dysfunction syndrome (MODS) comprise a heterogeneous population, which complicates research in its pathogenesis. Elucidation of the mechanisms involved in the development of MODS will ultimately necessitate the collection of tissue samples and the performance of invasive procedures. These requirements greatly reduce the possibilities for research in human subjects. Therefore, an animal model for MODS is a necessary and valuable tool. In the mid 1980s, the zymosan-induced generalized inflammation (ZIGI) model was introduced. Intraperitoneal injection of zymosan in mice or rats leads, in the course of 1 to 2 weeks, to increasing organ damage and dysfunction. The ZIGI model has been recognized as the one that best resembles human MODS and it has been used widely to study systemic inflammation in relation to organ failure. This review describes the ZIGI model and gives an overview of the results obtained.

Pentoxifylline does not improve outcome in a murine model for the multiple-organ dysfunction syndrome.

OBJECTIVE: To investigate the effects of pentoxifylline (PTX) administration in a murine model for the multiple-organ dysfunction syndrome (MODS). DESIGN AND SETTING: Prospective double-blind randomized animal study in a university research laboratory. INTERVENTIONS AND MEASUREMENTS: Sixty C57BL/6 mice were given an aseptic intraperitoneal injection of lipopolysaccharide followed after 6 days by zymosan (day 0) at a dose of either 0.9 or 1.0 mg/g body weight. Starting on day 0 mice were administered PTX at a dose of 80 mg/kg body weight or saline per os every 8 h. On day 17 surviving animals were killed, and their liver, lungs, spleen, and kidneys were collected. RESULTS: Mortality, course of body temperature, body weight, and macroscopic lung damage were similar between zymosan-treated groups. Administration of PTX did not significantly alter survival, body temperature, body weight, or macroscopic lung damage. In addition, there were no significant differences in organ weights between mice that received PTX and mice that received PBS. Although PTX inhibited the lipopolysaccharide-induced increase in tumor necrosis factor alpha and interleukin 6 expression (but not interleukin 1beta expression) at both mRNA and protein level in a murine macrophage cell line, tumor necrosis factor alpha mRNA expression in the livers of PTX-treated mice was not significantly inhibited. CONCLUSIONS: The results reported here do not support the hypothesis that PTX improves outcome in zymosan-induced multiple-organ dysfunction in mice.

Leukocytes in Complex Regional Pain Syndrome type I.

OBJECTIVE: The pathophysiology of Complex Regional Pain Syndrome type I (CRPS I) is unclear. An inflammatory reaction may cause the syndrome in which leukocytes may play an important role. MATERIALS AND METHODS: In this pilot study of six patients with acute warm CRPS I, we performed radiolabeled autologous leukocyte scans of both hands, in order to assess leukocyte accumulation. Comparison was made with the unaffected limb, and with three control patients with a Colles fracture without CRPS I. RESULTS: Images of the CRPS I patients obtained 4 h after leukocyte injection provided the clearest results. At 4 h post-injection, there was clear, asymmetrical leukocyte accumulation in the affected extremity with a mean ratio of 1.49+/-0.19. In control patients, no asymmetry was observed between hands (mean ratio 1.09+/-0.06), indicating the absence of specific leukocyte accumulation. There was a statistically significant difference between CRPS I and control subjects 4 h post injection (p=0.012). CONCLUSION: We found a significantly increased accumulation of leukocytes in patients with CRPS I. This is the first study to show a possible role for leukocytes in the pathophysiology of acute CRPS I.

Advanced trauma life support study: quality of diagnostic and therapeutic procedures.

BACKGROUND: The introduction of the ATLS course in The Netherlands in 1995 provided for an opportunity to compare data of trauma patients between a pre-ATLS and a post-ATLS period. MATERIALS AND METHODS: Over a 3-year period (May 1996 - September 1997 pre ATLS; December 1997-April 1999 post ATLS) 63 trauma patients with an AIS-ISS > or = 16 (n = 31, pre-ATLS and n = 32, post-ATLS) were prospectively studied in two community residency training (ACS Level III) hospitals. All diagnostic and therapeutic procedures were recorded by a video-camera and evaluated by a neutral faculty of six experienced ATLS trained specialists. RESULTS: Ten out of 14 interventions were performed qualitatively better in the post-ATLS group, while also the overall score was highly significantly better (4.2 pre-ATLS and 5.8 post-ATLS, p < 0.0001). CONCLUSION: Using the opinion of an expert team, this study identified a significantly lower number of patients with inadequate management.

Increased expression of matrix metalloproteinases in the murine zymosan-induced multiple organ dysfunction syndrome.

Matrix metalloproteinases (MMPs) have been implicated as mediators of tissue damage in several inflammatory diseases. Since the multiple organ dysfunction syndrome (MODS) is thought to result from systemic inflammation, overactivation of MMPs could contribute to the organ damage observed. The expression and activity of several MMPs were studied in a murine model for MODS. Sixty mice were given an aseptic intraperitoneal injection of lipopolysaccharide, followed, after 6 days, by zymosan. At days 2, 5, 8, 12, and 16 after the injection of zymosan, the liver, lungs, spleen, and kidneys were collected from groups of mice for either RNA extraction, gelatinase zymography and collagenase (MMP-1 and -13) assays (six mice per time point), or immunohistochemistry (three mice per time point). A group of nine mice did not receive zymosan and acted as controls. The expression of MMP-2 mRNA in zymosan-treated mice was strongly up-regulated in liver tissue only. For MMP-9, this was the case in all organs examined. Quantitative gelatin zymography demonstrated the near complete absence of any gelatinase activity in tissues from control mice. However, in the liver, lungs, and especially the spleen of zymosan-treated animals, significantly increased activity of proform and active MMP-2 and -9 was observed with time. Overall, MMP-1 and -13 activities were very low in all samples from the liver and lungs. In the spleen, however, high levels of MMP-1 and -13 were observed in zymosan-treated animals. Immunohistochemical staining for MMP-2 was detected in the liver and spleen, but not in lung and kidney tissue of zymosan-treated animals. Staining for MMP-9 could be detected in liver, lung, and spleen tissues of zymosan-treated mice. For both MMPs, staining appeared to be limited to phagocytes. In conclusion, the data suggest a role for MMPs, especially MMP-9, in the pathogenesis of MODS.

Tissue- and time-dependent upregulation of cytokine mRNA in a murine model for the multiple organ dysfunction syndrome.

OBJECTIVE: We sought to quantitate the course of specific cytokine mRNA expression in tissues that exhibit increasing histopathological changes in time in an animal model for the multiple organ dysfunction syndrome (MODS). SUMMARY BACKGROUND DATA: The development of treatment protocols for MODS requires elucidation of the mechanisms and mediators involved. To devise logical interventions, it is necessary to collect data on cytokine expression at tissue level during the development of MODS. METHODS: Ninety-four C57BL/6 mice were given an intraperitoneal injection of 40 microg of lipopolysaccharide (LPS), followed by zymosan at a dose of 0.8 mg/g body weight 6 days later (day 0). Six additional animals did not receive zymosan and acted as controls. At several time points after zymosan injection, 6 randomly assigned, zymosan-treated animals were killed, and their livers, lungs, spleens, and kidneys were collected. mRNA expression of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, macrophage migration inhibiting factor, IL-12, interferon-gamma, and IL-10 was measured using a real-time reverse transcription-polymerase chain reaction assay. RESULTS: The injection of zymosan induced an acute peritonitis, followed by an apparent recovery. From approximately day 6 onwards, animals started to display MODS-like symptoms. During the peritonitis phase, up-regulation of cytokine mRNA was limited. During the period of apparent recovery, cytokine mRNA expression strongly increased, mostly reaching its maximum at day 9 when deterioration of the clinical condition had already set in. The up-regulation of tumor necrosis factor-alpha mRNA was most pronounced, especially in the lungs and liver. CONCLUSIONS: Interventions should preferentially be targeted against multiple cytokines and, at least in this model, there may be a treatment window well after the initial challenge.

Clinical impact of advanced trauma life support.

The Advanced Trauma Life Support (ATLS) course sponsored by the American College of Surgeons Committee On Trauma (ACSCOT) presents a standardized method of initial trauma care. This study attempted to measure any changes in morbidity and mortality in trauma patients after the introduction of ATLS training. Over a 3-year period (May 1996 to September 1997-pre-ATLS period; December 1997 to April 1999-post-ATLS period), 63 trauma patients with an Injury Severity Scale (ISS) > or =16 (n = 31, pre-ATLS and n = 32, post-ATLS) were prospectively studied in two community teaching hospitals. There was no significant difference in mortality rate between groups (48% [15 of 31] pre-ATLS vs. 30% [10 of 32] post-ATLS; P = .203, Fisher exact test). Mortality rates within the ISS range of 16 to 25 were 64% (nine of 14 pre-ATLS) versus 29% (five of 17 post-ATLS), and for the ISS 26 to 35 subgroup, 40% (four of 10 pre-ATLS) versus 25% (two of eight post-ATLS), and within the ISS 36 to 75 subgroup, 29% (two of seven pre-ATLS) versus 43% (three of seven post-ATLS). There was a significant difference in mortality during the first 60 minutes after admission: 0.0% post-ATLS versus 24.2% pre-ATLS (P = .002, Fisher exact test (95% confidence interval ranged from 12-45% in the pre-ATLS group and 0-11% in the post-ATLS group). According to the TRISS methodology (a worldwide-accepted mathematical method to calculate chances of survival through logistical regression),ATLS improved outcome from sub-"Major Trauma Outcome Study" (MTOS) standard results (z = -2.9 to a MTOS standard result z = -0.49). Our data demonstrate that introduction of the ATLS program significantly improved trauma patient outcome in the first hour after admission, as well as improvement from sub-MTOS standard to MTOS standard levels.

Capillary blood gas analysis in complex regional pain syndrome: a pilot study.

BACKGROUND: The pathophysiology of complex regional pain syndrome type 1 (CRPS 1) is still a matter of debate. An inflammatory reaction may cause the syndrome. Increasing evidence points to a role for impairment of oxygen metabolism in the affected limb. METHODS: In this pilot study (16 patients) we performed capillary blood gas analysis in extremities with acute CRPS 1, in order to assess oxygen saturation and lactate concentrations. Comparison was made with the unaffected limb for capillary blood pH, pO(2), SaO(2), and lactate and glucose concentrations. RESULTS: No statistically significant differences could be found. CONCLUSIONS: Capillary blood gas analysis is not useful to detect changes in oxygen saturation and lactate concentrations in CRPS 1.

Trauma resuscitation time.

Documenting the timing and organisation of trauma resuscitation can be utilised to assess performance standards, and to ensure a high quality of trauma resuscitation procedures. Since there is no European literature available on trauma resuscitation time (TRT) in the emergency room, the aim of this descriptive study is to evaluate TRT in the Netherlands. The introduction of an ATLS-trained prehospital mobile medical team (MMT) in the Nijmegen area initiated the on-site advanced trauma life-support for the prehospital management of trauma patients. We studied TRT in two groups of patients, one with, the other without on-site care by a MMT. In the emergency room the use of videotape recording was chosen to document trauma resuscitation (22 actions) and TRT. A specially flow-chart was used to define the TRT-procedures. We studied 43 patients; 27 without MMT treatment and 16 with MMT treatment. The activities were divided into the ABCDE's of trauma care. Significant more patients of the MMT group were intubated before arrival in the hospital (12/16 (75%) versus 2/27 (2%), P<0.05). Eleven definitive airway management interventions (intubation) and one thoracic drainage in the non-MMT group were demanded by the protocol, but not performed before arrival in the hospital. Sixteen out of 22 actions that were documented were carried out at an earlier stage in the MMT group. There was no significant difference between the resuscitation times; in both groups the recorded median time was approximately 43 min. This prospective analysis demonstrates the timing of resuscitation procedures in a resuscitation room and provides some insight into the timing of ATLS initial assessment.