Radim Jančálek

Bilateral changes of TNF-α and IL-10 protein in the lumbar and cervical dorsal root ganglia following a unilateral chronic constriction injury of the sciatic nerve

BackgroundThere is a growing body of evidence that unilateral nerve injury induces bilateral response, the mechanism of which is not exactly known. Because cytokines act as crucial signaling molecules for response of peripheral nerves to injury, they may be induced to mediate the reaction in remote structures.MethodsWe studied levels of tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) proteins using ELISA in the ipsilateral and contralateral lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) from naïve rats, rats operated on to create unilateral chronic constriction injury (CCI) of the sciatic nerve, and sham-operated rats. Withdrawal thresholds for mechanical allodynia and thermal hyperalgesia were measured in the ipsilateral and contralateral hind and forepaws.ResultsThe ipsilateral hind paws of all rats operated upon for CCI displayed decreased withdrawal thresholds for mechanical allodynia and thermal hyperalgesia, while no significant behavioral changes were found in the contralateral hind paws and both forepaws. Significantly lower baseline levels of TNF-α and IL-10 protein were measured by ELISA in the lumbar than cervical DRG of naïve rats. Bilateral elevation of TNF-α was induced in both the lumbar and cervical DRG by unilateral CCI of the sciatic nerve for 7 and 14 days, while the level of IL-10 protein was increased bilaterally in the lumbar DRG 1 and 3 days after operation. IL-10 levels declined bilaterally even below baseline level in both cervical and lumbar DRG 7 days from CCI and normalized after 14 days. In contrast to no significant changes in TNF-α, level of IL-10 protein was significantly increased in the ipsilateral lumbar DRG after 3 days and bilaterally in the lumbar DRG after 14 days from sham operation.ConclusionsThe results of our experiments show a bilateral elevation of TNF-α and IL-10 not only in the homonymous DRG but also in the heteronymous DRG unassociated with the injured nerve. This suggests that bilaterally increased levels of TNF-α and IL-10 in DRG following unilateral CCI are linked with general neuroinflammatory reaction of the nervous system to injury rather than only to development and maintenance of neuropathic pain.

Potential of MR spectroscopy for assessment of glioma grading.

BACKGROUND Magnetic resonance spectroscopy (MRS) is an imaging diagnostic method based that allows non-invasive measurement of metabolites in tissues. There are a number of metabolites that can be identified by standard brain proton MRS but only a few of them has a clinical significance in diagnosis of gliomas including N-acetylaspartate, choline, creatine, myo-inositol, lactate, and lipids. METHODS In this review, we describe potential of MRS for grading of gliomas. RESULTS Low-grade gliomas are generally characterized by a relatively high concentration of N-acetylaspartate, low level of choline and absence of lactate and lipids. The increase in creatine concentration indicates low-grade gliomas with earlier progression and malignant transformation. Progression in grade of a glioma is reflected in the progressive decrease in the N-acetylaspartate and myo-inositol levels on the one hand and elevation in choline level up to grade III on the other. Malignant transformation of the glial tumors is also accompanied by the presence of lactate and lipids in MR spectra of grade III but mainly grade IV gliomas. It follows that MRS is a helpful method for detection of glioma regions with aggressive growth or upgrading due to favorable correlation of the choline and N-acetylaspartate levels with histopathological proliferation index Ki-67. Thus, magnetic resonance spectroscopy is also a suitable method for the targeting of brain biopsies. CONCLUSIONS Gliomas of each grade have some specific MRS features that can be used for improvement of the diagnostic value of conventional magnetic resonance imaging in non-invasive assessment of glioma grade.

Intra- and Extraneuronal Changes of Immunofluorescence Staining for TNF- and TNFR1 in the Dorsal Root Ganglia of Rat Peripheral Neuropathic Pain Models

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-α and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury.2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-α and TNFR1 proteins. The TNF-α and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-α and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-α and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury.3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-α and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFα IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-α after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFα IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites.4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF.5. Our results suggest more sources of TNF-α protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-α molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-α effect on the primary sensory neurons can be mediated following nerve injury.

Why and how to spare the hippocampus during brain radiotherapy: the developing role of hippocampal avoidance in cranial radiotherapy

The goal of this review is to summarize the rationale for and feasibility of hippocampal sparing techniques during brain irradiation. Radiotherapy is the most effective non-surgical treatment of brain tumors and with the improvement in overall survival for these patients over the last few decades, there is an effort to minimize potential adverse effects leading to possible worsening in quality of life, especially worsening of neurocognitive function. The hippocampus and associated limbic system have long been known to be important in memory formation and pre-clinical models show loss of hippocampal stem cells with radiation as well as changes in architecture and function of mature neurons. Cognitive outcomes in clinical studies are beginning to provide evidence of cognitive effects associated with hippocampal dose and the cognitive benefits of hippocampal sparing. Numerous feasibility planning studies support the feasibility of using modern radiotherapy systems for hippocampal sparing during brain irradiation. Although results of the ongoing phase II and phase III studies are needed to confirm the benefit of hippocampal sparing brain radiotherapy on neurocognitive function, it is now technically and dosimetrically feasible to create hippocampal sparing treatment plans with appropriate irradiation of target volumes. The purpose of this review is to provide a brief overview of studies that provide a rationale for hippocampal avoidance and provide summary of published feasibility studies in order to help clinicians prepare for clinical usage of these complex and challenging techniques.

Increased invasion of ED-1 positive macrophages in both ipsi- and contralateral dorsal root ganglia following unilateral nerve injuries

There is an increasing evidence that unilateral nerve injury induces cellular and molecular changes in the associated DRG not only on the ipsilateral but also in the contralateral side. In this investigation, ED-1+ macrophages were quantified by image analysis in the naïve L5 DRG (nDRG) and compared with the ipsi- and contralateral ones 2 and 4 weeks after unilateral sciatic nerve ligature and ventral root transection (VRT). A few ED-1+ macrophages were found in nDRG but not closely associated with the neuronal bodies. In contrast, following nerve injuries ED-1+ macrophages and their processes were frequently located close neuronal bodies and became their satellite cells. Moreover, an increased number of ED-1+ cells was found in the ipsilateral DRG 2 weeks after unilateral sciatic nerve ligature or VRT, but no significant differences were measured between 2 and 4 weeks after both types of nerve lesion. Contralateral DRG displayed a significant enhanced number of ED-1+ cells no sooner than 4 weeks from sciatic nerve ligature. In contrast, VRT induced a significant increased invasion of the ED-1+ cells in the contralateral DRG as early as 2 weeks after operation. Our experiments indicate that a significantly higher number of ED-1+ macrophages remained in both ipsi- and contralateral DRG up to 4 weeks from nerve injury. Based on results from different models of nerve injury, we suggest that more than one mechanism operates to stimulate the invasion of ED-1+ macrophages into the DRG including retrograde transport of factors produced during Wallerian degeneration or their delivery by blood flow. Signaling for macrophage invasion into DRG contralateral to nerve injury may be mediated by lost motoneurons or by interneurones.

Role of inflammation and cytokines in peripheral nerve regeneration.

This chapter provides a review of immune reactions involved in classic as well as alternative methods of peripheral nerve regeneration, and mainly with a view to understanding their beneficial effects. Axonal degeneration distal to nerve damage triggers a cascade of inflammatory events alongside injured nerve fibers known as Wallerian degeneration (WD). The early inflammatory reactions of WD comprise the complement system, arachidonic acid metabolites, and inflammatory mediators that are related to myelin fragmentation and activation of Schwann cells. Fine-tuned upregulation of the cytokine/chemokine network by Schwann cells activates resident and hematogenous macrophages to complete the clearance of axonal and myelin debris and stimulate regrowth of axonal sprouts. In addition to local effects, immune reactions of neuronal bodies and glial cells are also implicated in the survival and conditioning of neurons to regenerate severed nerves. Understanding of the cellular and molecular interactions between the immune system and peripheral nerve injury opens new possibilities for targeting inflammatory mediators to improve functional reinnervation.

Bilateral changes of IL-10 protein in lumbar and cervical dorsal root ganglia following proximal and distal chronic constriction injury of peripheral nerve

Interleukin-10 prevents transition of a physiological inflammatory reaction to a pathological state that may result in neuropathic pain. We studied bilateral changes of IL-10 protein levels in L4-L5 and C7-C8 dorsal root ganglia (DRG) after a chronic constriction injury (CCI) of either L4-L5 spinal nerves (pCCI) or the sciatic nerve (dCCI). Rats undergoing pCCI or dCCI were left to survive for 1, 3, 7 or 14 d, sham-operated rats for 3 or 14 d. After the survival time, C7-C8 and L4-L5 DRG were removed bilaterally from naïve, operated, and sham-operated rats and IL-10 protein was detected by immunohistochemical staining and measured using ELISA analysis. Unilateral pCCI and dCCI induced a transient bilateral elevation in IL-10 protein level not only in the homonymous lumbar DRG but also in the heteronymous cervical DRG nonassociated with the spinal segments of constricted nerve. Sham operations also induced bilateral elevation of IL-10 protein in both homonymous and heteronymous DRG. Our experiments revealed that the more proximal is a nerve injury the more rapid is the initial increase and slower the subsequent decrease of IL-10 protein level in DRG. Changes of IL-10 protein in DRG nonassociated with damaged nerve could be related to a general neuroinflammatory reaction of the nervous system to injury and thereby promote potential of the DRG neurons for regenerating their axons following a conditioning lesion.

Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar Microdiscectomy: A Prospective Cross-Sectional Study

Background The relationship between intervertebral disc degeneration and chronic infection by Propionibacterium acnes is controversial with contradictory evidence available in the literature. Previous studies investigating these relationships were under-powered and fraught with methodical differences; moreover, they have not taken into consideration P. acnes’ ability to form biofilms or attempted to quantitate the bioburden with regard to determining bacterial counts/genome equivalents as criteria to differentiate true infection from contamination. The aim of this prospective cross-sectional study was to determine the prevalence of P. acnes in patients undergoing lumbar disc microdiscectomy. Methods and Findings The sample consisted of 290 adult patients undergoing lumbar microdiscectomy for symptomatic lumbar disc herniation. An intraoperative biopsy and pre-operative clinical data were taken in all cases. One biopsy fragment was homogenized and used for quantitative anaerobic culture and a second was frozen and used for real-time PCR-based quantification of P. acnes genomes. P. acnes was identified in 115 cases (40%), coagulase-negative staphylococci in 31 cases (11%) and alpha-hemolytic streptococci in 8 cases (3%). P. acnes counts ranged from 100 to 9000 CFU/ml with a median of 400 CFU/ml. The prevalence of intervertebral discs with abundant P. acnes (≥ 1x103 CFU/ml) was 11% (39 cases). There was significant correlation between the bacterial counts obtained by culture and the number of P. acnes genomes detected by real-time PCR (r = 0.4363, p<0.0001). Conclusions In a large series of patients, the prevalence of discs with abundant P. acnes was 11%. We believe, disc tissue homogenization releases P. acnes from the biofilm so that they can then potentially be cultured, reducing the rate of false-negative cultures. Further, quantification study revealing significant bioburden based on both culture and real-time PCR minimize the likelihood that observed findings are due to contamination and supports the hypothesis P. acnes acts as a pathogen in these cases of degenerative disc disease.

The Diagnostic Ability of Follow-Up Imaging Biomarkers after Treatment of Glioblastoma in the Temozolomide Era: Implications from Proton MR Spectroscopy and Apparent Diffusion Coefficient Mapping.

Objective. To prospectively determine institutional cut-off values of apparent diffusion coefficients (ADCs) and concentration of tissue metabolites measured by MR spectroscopy (MRS) for early differentiation between glioblastoma (GBM) relapse and treatment-related changes after standard treatment. Materials and Methods. Twenty-four GBM patients who received gross total resection and standard adjuvant therapy underwent MRI examination focusing on the enhancing region suspected of tumor recurrence. ADC maps, concentrations of N-acetylaspartate, choline, creatine, lipids, and lactate, and metabolite ratios were determined. Final diagnosis as determined by biopsy or follow-up imaging was correlated to the results of advanced MRI findings. Results. Eighteen (75%) and 6 (25%) patients developed tumor recurrence and pseudoprogression, respectively. Mean time to radiographic progression from the end of chemoradiotherapy was 5.8 ± 5.6 months. Significant differences in ADC and MRS data were observed between those with progression and pseudoprogression. Recurrence was characterized by N-acetylaspartate ≤ 1.5 mM, choline/N-acetylaspartate ≥ 1.4 (sensitivity 100%, specificity 91.7%), N-acetylaspartate/creatine ≤ 0.7, and ADC ≤ 1300 × 10−6 mm2/s (sensitivity 100%, specificity 100%). Conclusion. Institutional validation of cut-off values obtained from advanced MRI methods is warranted not only for diagnosis of GBM recurrence, but also as enrollment criteria in salvage clinical trials and for reporting of outcomes of initial treatment.

Signaling mechanisms in mirror image pain pathogenesis.

It is now clear that a peripheral nerve lesion affects contralateral non-lesioned structures, and thus such a lesion can result in mirror image pain. The pathogenesis is still not exactly known, but there are some possible signaling pathways in the contralateral reaction of the nerve tissue after unilateral nerve injury. Potential signaling pathways of contralateral changes can be generally divided into humoral and neuronal mechanisms. Damage to peripheral nerves or spinal roots produces a number of breakdown products with development of an aseptic inflammatory reaction. Released immunomodulatory cytokines are believed to be transported via blood or cerebrospinal fluid into the contralateral part of the body affecting spinal roots, dorsal root ganglia or peripheral nerves. Because neurons are elements of a highly organized network, injury to the peripheral neuron results in signals that travel transneuronally into the central nervous system and affects the contralateral homonymous neurons. There is also evidence that spinal glia creates and maintain pathological pain. Additionally, there may be compensatory changes in behavior of animals with an impact on contralateral neurons, such as altered stance and motor performance or autonomic reflex changes. Although the transneuronal signaling pathway appears to be plausible, the humoral signaling pathway or other communication systems cannot be excluded at this time. Knowledge about these processes has clinical implications for the understanding of chronic neuropathic pain states, and, therefore, further studies will be necessary. Understanding signaling mechanisms in mirror image pain pathogenesis may provide novel therapeutic targets for the management of neuropathic pain.