Switching Rat Resident Macrophages from M1 to M2 Phenotype by Iba1 Silencing Has Analgesic Effects in SNL-Induced Neuropathic Pain.

Resident macrophages from dorsal root ganglia are important for the development of traumatic-induced neuropathic pain. In the first 5-7 days after a traumatic sciatic nerve injury (i.e., spinal nerve ligation (SNL), spared nerve injury (SNI), sciatic nerve transection or sciatic nerve ligation and transection), Ionized binding adapter protein 1 (Iba1) (+) resident macrophages cluster around dorsal root ganglia neurons, possibly contributing to nerve injury-induced hypersensitivity. Since infiltrating macrophages gradually recruited to the lesion site peak at about 7 days, the first few days post-lesion offer a window of opportunity when the contribution of Iba1 (+) resident macrophages to neuropathic pain pathogenesis could be investigated. Iba1 is an actin cross-linking cytoskeleton protein, specifically located only in macrophages and microglia. In this study, we explored the contribution of rat Iba1 (+) macrophages in SNL-induced neuropathic pain by using intra-ganglionic injections of naked Iba1-siRNA, delivered at the time the lesion occurred. The results show that 5 days after Iba1 silencing, Iba1 (+) resident macrophages are switched from an M1 (pro-inflammatory) phenotype to an M2 (anti-inflammatory) phenotype, which was confirmed by a significant decrease of M1 markers (CD32 and CD86), a significant increase of M2 markers (CD163 and Arginase-1), a reduced secretion of pro-inflammatory cytokines (IL-6, TNF-α and IL-1ß) and an increased release of pro-regenerative factors (BDNF, NGF and NT-3) which initiated the regrowth of adult DRG neurites and reduced SNL-induced neuropathic pain. Our data show for the first time, that it is possible to induce macrophages towards an anti-inflammatory phenotype by interacting with their cytoskeleton.

Implantable, Programmable, and Wireless Device for Electrical Stimulation of the Dorsal Root Ganglion in Freely-Moving Rats: A Proof of Concept Study.

OBJECTIVE: This was a proof of concept study, based on systematic reviews of the efficacy and safety of the dorsal root ganglion (DRG) stimulation. The main objective was to develop an implantable, programmable, and wireless device for electrical stimulation of DRG and a methodology that can be used in translational research, especially to understand the mechanism of neuromodulation and to test new treatment modalities in animal models of pain. METHODS: We developed and tested a stimulator that uses a battery-powered microelectronic circuit, to generate constant current square biphasic or monophasic pulsed waveform of variable amplitudes and duration. It is controlled by software and an external controller that allows radio frequency communication with the stimulator. The stimulator was implanted in Sprague-Dawley (SD) rats. The lead was positioned at the L5 DRG level, while the stimulator was placed in the skin pocket at the ipsilateral side. Forty-five animals were used and divided into six groups: spinal nerve ligation (SNL), chronic compression injury of the DRG (CCD), SNL + active DRG stimulation, intact control group, group with the implanted sham stimulator, and sham lead. Behavioral testing was performed on the day preceding surgery and three times postoperatively (1st, 3rd, and 7th day). RESULTS: In animals with SNL, neurostimulation reduced pain-related behavior, tested with pinprick hyperalgesia, pinprick withdrawal test, and cold test, while the leads per se did not cause DRG compression. The rats well tolerated the stimulator. It did not hinder animal movement, and it enabled the animals to be housed under regular conditions. CONCLUSION: A proof-of-concept experiment with our stimulator verified the usability of the device. The stimulator enables a wide range of research applications from adjusting stimulation parameters for different pain conditions, studying new stimulation methods with different frequencies and waveforms to obtain knowledge about analgesic mechanisms of DRG stimulation.

Efficacy and Safety of Pulsed Radiofrequency as a Method of Dorsal Root Ganglia Stimulation in Patients with Neuropathic Pain: A Systematic Review.

OBJECTIVE: Pulsed radiofrequency (PRF) is a nonablative pain treatment that uses radiofrequency current in short high-voltage bursts, resulting in interruption of nociceptive afferent pathways. We conducted a systematic review with the aim to create a synthesis of evidence about the efficacy and safety of PRF applied to the dorsal root ganglion (DRG) for the treatment of neuropathic pain. METHODS: We searched MEDLINE, CINAHL, Embase, and PsycINFO through January 8, 2019, as well as ClinicalTrials.gov and the clinical trial register of the World Health Organization. All study designs were eligible. We assessed risk of bias using the Cochrane tool for randomized controlled trials and the Risk Of Bias In Non-Randomized Studies of Interventions (ROBINS-I). We assessed level of evidence using the Oxford tool and quality of evidence with GRADE. RESULTS: We included 28 studies with participants suffering from lumbosacral, cervical, or thoracic radicular pain, post-herpetic neuralgia, neuropathicbone pain in cancer patients, or carpal tunnel syndrome. Only five studies were randomized controlled trials (RCTs), while others were of nonrandomized designs, predominantly before and after comparisons. A total of 991 participants were included, with a median number (range) of 31 (1-101) participants. Only 204 participants were included in the RCTs, with a median number (range) of 38 (23-62) participants. The overall quality of evidence was low, as the majority of the included studies were rated as evidence level 4 or 5. The quality of evidence was very low. CONCLUSIONS: Evidence about the efficacy and safety of PRF of the DRG for the treatment of neuropathic pain is based mainly on results from very small studies with low evidence quality. Current research results about the benefits of PRF of the DRG for the treatment of neuropathic pain should be considered preliminary and confirmed in high-quality RCTs with sufficient numbers of participants.

Efficacy and safety of pulsed radiofrequency as a method of dorsal root ganglia stimulation for treatment of non-neuropathic pain: a systematic review.

BACKGROUND: We systematically reviewed the evidence on the efficacy and safety of dorsal root ganglion (DRG) targeted pulsed radiofrequency (PRF) versus any comparator for treatment of non-neuropathic pain. METHODS: We searched MEDLINE, CINAHL, Embase, PsycINFO, clinicaltrials.gov and WHO clinical trial register until January 8, 2019. All study designs were eligible. Two authors independently conducted literature screening. Primary outcomes were pain intensity and serious adverse events (SAEs). Secondary outcomes were any other pain-related outcome and any other safety outcome that was reported. We assessed the risk of bias using the Cochrane tool and Risk of Bias In Non-randomized Studies of Interventions (ROBINS-I). We conducted narrative evidence synthesis and assessed the conclusiveness of included studies regarding efficacy and safety. RESULTS: We included 17 studies with 599 participants, which analyzed various pain syndromes. Two studies were randomized controlled trials; both included participants with low back pain (LBP). Non-randomized studies included patients with the following indications: LBP, postsurgical pain, pain associated with herpes zoster, cervicogenic headache, complex regional pain syndrome type 1, intractable vertebral metastatic pain, chronic scrotal and inguinal pain, occipital radiating pain in rheumatoid arthritis and chronic migraine. In these studies, the PRF was usually initiated after other treatments have failed. Eleven studies had positive conclusive statements (11/17) about efficacy; the remaining had positive inconclusive statements. Only three studies provided conclusiveness of evidence statements regarding safety - two indicated that the evidence was positive conclusive, and one positive inconclusive. The risk of bias was predominantly unclear in randomized and serious in non-randomized studies. CONCLUSION: Poor quality and few participants characterize evidence about benefits and harms of DRG PRF in patients with non-neuropathic pain. Results from available studies should only be considered preliminary. Not all studies have reported data regarding the safety of the intervention, but those that did, indicate that the intervention is relatively safe. As the procedure is non-destructive and early results are promising, further comparative studies about PRF in non-neuropathic pain syndromes would be welcomed.

Overactive bladder symptoms in patients undergoing rigid and flexible cystoscopy.

PURPOSE: To evaluate overactive bladder (OAB) symptoms in patients undergoing diagnostic cystoscopy. Overall changes in the entire study population were assessed, as well as broken down by various subgroups. METHODS: A prospective multi-center study among consecutive 450 adults undergoing diagnostic cystoscopy was conducted. OAB-symptoms were evaluated with the validated eight-item OAB Screening Awareness Tool (OAB-V8) immediately before and on days 1, 4, and 7 after cystoscopy. Patients were distinguished between being OAB-negative and OAB-positive (< 8 and ≥ 8 sum-score, respectively). Average sum-scores and subdomains were evaluated. RESULTS: Before cystoscopy, 44.7% of patients were screened OAB-positive and 55.3% OAB-negative. Out of those being screened negative, development of de-novo OAB was noticed in 16.8%, declining to 8.1% on day 7 (p < 0.001). In patients being OAB-positive before cystoscopy, a decline of OAB-positivity was noted during follow-up (p < 0.001). No statistically significant differences were noted when broken down by gender (p = 0.92), age (p = 0.82) and type cystoscope (rigid vs. flexible, p = 0.38). Average sum-scores declined from 8.68 before cystoscopy to 6.9 during follow-up. Flexible cystoscopy was superior over rigid in four subdomains: uncomfortable urge to urinate (p = 0.04), sudden urge to urinate with little or no warning (p = 0.02), uncontrollable urge to urinate (p = 0.03), and urine loss associated with a strong desire to void (p = 0.009). CONCLUSION: OAB-symptoms are common in patients undergoing cystoscopy. Cystoscopy itself can cause de-novo OAB-symptoms. Controversially, a decline of OAB-symptoms was noted after cystoscopy when patients were screened OAB-positive before cystoscopy. Flexible scopes were superior in some subdomains.

Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0µM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.

Intrathecal inhibition of calcium/calmodulin-dependent protein kinase II in diabetic neuropathy adversely affects pain-related behavior.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered an important enzyme contributing to the pathogenesis of persistent pain. The aim of this study was to test whether intrathecal injection of CaMKII inhibitors may reduce pain-related behavior in diabetic rats. Male Sprague-Dawley rats were used. Diabetes was induced with intraperitoneal injection of 55mg/kg streptozotocin. Two weeks after diabetes induction, CaMKII inhibitor myristoil-AIP or KN-93 was injected intrathecally. Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2h and 24h after the intrathecal injection. The expression of total CaMKII and its alpha isoform in dorsal horn was quantified using immunohistochemistry. Intrathecal injection of mAIP and KN-93 resulted in significant decrease in expression of total CaMKII and CaMKII alpha isoform activity. Also, mAIP and KN93 injection significantly increased sensitivity to a mechanical stimulus 24h after i.t. injection. Intrathecal inhibition of CaMKII reduced the expression of total CaMKII and its CaMKII alpha isoform activity in diabetic dorsal horn, which was accompanied with an increase in pain-related behavior. Further studies about the intrathecal inhibition of CaMKII should elucidate its role in nociceptive processes of diabetic neuropathy.

Intraganglionic AAV6 results in efficient and long-term gene transfer to peripheral sensory nervous system in adult rats.

We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10(9) viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.

Adherence to pharmacological treatment of chronic nonmalignant pain in individuals aged 65 and older.

BACKGROUND: Medication nonadherence is a frequent problem in the treatment of chronic conditions. OBJECTIVE: To study the adherence to pharmacological treatment of chronic nonmalignant pain, as well as factors and patient attitudes related to nonadherence in patients aged ≥65 years. METHODS: The cross-sectional study was conducted with a self-administered questionnaire among 100 patients aged ≥65 years by five family physicians at the Health Care Centre Mostar, Bosnia and Herzegovina. RESULTS: According to their own statements, 57% of the patients were nonadherent, while 84% exhibited some form of nonadherence on the Morisky scale. The patients reported a mean pain intensity of 6.6 ± 2.2 on a visual analog scale. The most common deviation from the prescribed therapy was self-adjustment of the dose and medical regimen based on the severity of pain. Polymedication correlated positively with nonadherence. Nonsteroidal anti-inflammatory drugs were the most frequently prescribed medications. The majority of the participants (59%) believed that higher pain intensity indicates progression of the disease, and half of the participants believed that one can easily become addicted to pain medications. Nonadherence was associated with patient attitudes about addiction to analgesics and ability of analgesics to control pain. CONCLUSION.: High pain intensity and nonadherence found in this study suggest that physicians should monitor older patients with chronic nonmalignant pain more closely and pay more attention to patients' beliefs regarding analgesics to ensure better adherence to pharmacological therapy.

Failure of action potential propagation in sensory neurons: mechanisms and loss of afferent filtering in C-type units after painful nerve injury.

The T-junction of sensory neurons in the dorsal root ganglion (DRG) is a potential impediment to action potential (AP) propagation towards the CNS. Using intracellular recordings from rat DRG neuronal somata during stimulation of the dorsal root, we determined that the maximal rate at which all of 20 APs in a train could successfully transit the T-junction (following frequency) was lowest in C-type units, followed by A-type units with inflected descending limbs of the AP, and highest in A-type units without inflections. In C-type units, following frequency was slower than the rate at which AP trains could be produced in either dorsal root axonal segments or in the soma alone, indicating that the T-junction is a site that acts as a low-pass filter for AP propagation. Following frequency was slower for a train of 20 APs than for two, indicating that a cumulative process leads to propagation failure. Propagation failure was accompanied by diminished somatic membrane input resistance, and was enhanced when Ca(2+)-sensitive K(+) currents were augmented or when Ca(2+)-sensitive Cl(-) currents were blocked. After peripheral nerve injury, following frequencies were increased in axotomized C-type neurons and decreased in axotomized non-inflected A-type neurons. These findings reveal that the T-junction in sensory neurons is a regulator of afferent impulse traffic. Diminished filtering of AP trains at the T-junction of C-type neurons with axotomized peripheral processes could enhance the transmission of activity that is ectopically triggered in a neuroma or the neuronal soma, possibly contributing to pain generation.

Postlaminectomy stabilization of the spine in a rat model of neuropathic pain reduces pain-related behavior.

STUDY DESIGN: Spine deformity and pain-related behavior after laminectomy with and without spine stabilization were investigated. OBJECTIVE: We tested hypothesis that spine stabilization after extensive laminectomy can prevent spine deformation and consequent pain-related behavior. SUMMARY OF BACKGROUND DATA: Various ablative procedures requiring laminectomy have been tested for prevention or reversal of pain-related behavior in studies using experimental animals. However, there is no precise description indicating how laminectomy should be performed. Lack of standardized surgical techniques makes it difficult to achieve uniformity of result reporting and to compare results of different research groups meaningfully. METHODS: To test our hypothesis, extensive laminectomy with and without spine stabilization was performed in Sprague-Dawley rats. U-shaped surgical wire was used for stabilization of the spine. A validated test of mechanical hyperalgesia was used to test the development of neuropathic pain behavior after surgery. Deformity of the spine was evaluated by calculating deviation from the central axis on radiographs obtained in anteroposterior projection. RESULTS: Surgical stabilization of the spine after laminectomy prevented development of spinal deformity. Laminectomy without stabilization induced hyperalgesia on the 8th and 15th days after surgery. Group with stabilized spine exhibited significant reduction in pain-related behavior on the 8th and 15th postoperative days compared with the group without stabilization. CONCLUSION: Surgical stabilization of the spine after laminectomy prevented development of spinal deformity and pain-related behavior. Our results suggest that spine stabilization procedure should be used in all experimental pain models in which laminectomy is performed.

Direct injection into the dorsal root ganglion: technical, behavioral, and histological observations.

Direct injection of agents into the dorsal root ganglia (DRGs) offers the opportunity to manipulate sensory neuron function at a segmental level to explore pathophysiology of painful conditions. However, there is no described method that has been validated in detail for such injections in adult rats. We have found that 2 µl of dye injected through a pulled glass pipette directly into the distal DRG, exposed by a minimal foraminotomy, produces complete filling of the DRG with limited extension into the spinal roots. Injection into the spinal nerve required 3 µl to achieve comparable DRG filling, produced preferential spread into the ventral root, and was accompanied by substantial leakage of injected solution from the injection site. Injections into the sciatic nerve of volumes up to 10 µl did not reach the DRG. Transient hypersensitivity to mechanical stimulation at threshold (von Frey) and noxious levels (pin) developed after 2 µl saline injection directly into the DRG that was in part attributable to the surgical exposure procedure alone. Only minimal astrocyte activation in the spinal dorsal horn was evident after DRG saline injections. Injection of adeno-associated virus (AAV) vector conveying green fluorescent protein (GFP) transgene resulted in expression as soon as 1 day after injection into the DRG, including fibers in the spinal dorsal horn and columns. AAV injection into the DRG produced additional thermal hypersensitivity and withdrawal from the stroke of a brush and compromised motor performance. These findings demonstrate a method for selective injection of agents into single DRGs for anatomically restricted actions.

Lidocaine injection into the rat dorsal root ganglion causes neuroinflammation.

BACKGROUND: Injury of a spinal nerve or dorsal root ganglion (DRG) during selective spinal nerve blocks is a potentially serious complication that has not been adequately investigated. Our hypothesis was that local anesthetic injection into these structures may result in an inflammatory response and hyperalgesia. METHODS: We evaluated inflammatory and behavioral responses after injection of 4 microL lidocaine or saline into the L5 spinal nerve or DRG of rats after partial laminectomy. Behavioral testing was performed before and after surgery to examine hyperalgesia in response to nociceptive mechanical stimulation of the foot. DRGs were harvested and stained, and rings of immunoreactive glial cells around neurons were counted. RESULTS: Animals demonstrated hyperalgesia on the ipsilateral paw up to 4 days after lidocaine injection into the DRG but not after injection into the spinal nerve. The number of glial fibrillary acid protein immunopositive glial cell rings, which represent activation of satellite cells, significantly increased in DRGs after injection of lidocaine into either the DRG or the spinal nerve. The number of glial fibrillary acid protein-positive cells in the lidocaine-injected group was significantly larger than in the saline-injected group. Sporadic OX-42 immunopositive cells, which represent activated microglia, were also seen in lidocaine-injected DRGs. Testing for Pan-T expression, which labels activated T lymphocytes, showed no positive cells. CONCLUSIONS: Lidocaine injection into the DRG may produce hyperalgesia, possibly due to activation of resident satellite glial cells. In a clinical setting, local anesthetic injection into the DRG should be avoided during selective spinal nerve blocks.

The extent of laminectomy affects pain-related behavior in a rat model of neuropathic pain.

One of the unresolved questions in neuropathic pain research is whether we can prevent or reverse mechanical hyperalgesia by rhizotomy or ganglionectomy. However, one of the obstacles in answering that question is lack of a standardized surgical procedure used in experimental ganglionectomy. We tested the hypothesis that laminectomy performed during ganglionectomy induces lumbar column deformity. We further examined whether spinal deformity is a source of pain-related behavior. Five conditions were studied. Fifth and sixth lumbar (L5 and L6) ganglionectomy were performed in rats using either minimal or extensive laminectomy technique. Two other groups had minimal and extensive laminectomy without ganglionectomies. A final control group had no surgery. Sensory responsiveness of the plantar aspect of the hind paw was repeatedly tested, and a plain radiograph in anteroposterior projection was made to assess the extent of deformity by measurement of deformity angles. Hyperalgesia resulted in groups with extensive laminectomy regardless of performance or absence of ganglionectomy, while in groups with minimal laminectomy there was no increase in pain-related behavior. Lateral deformity of the spine was observed in rats with or without ganglionectomy, confirming that laminectomy can produce deformity. The extent of deformity was more pronounced in rats exposed to the extensive laminectomy. Our results indicate that laminectomy can produce spine deformity and that there is a direct relationship between the extent of laminectomy and the development of mechanical hypersensitivity. The data presented suggest that there is a need for standardization of laminectomy procedure in rat experimental pain models.

Developmental patterns of caspase-3, bax and bcl-2 proteins expression in the human spinal ganglia.

The distribution of the bcl-2, bax and caspase-3 proteins was investigated in the cells of developing human spinal ganglia. Paraffin sections of 10 human conceptuses between 5th and 9th gestational weeks were analysed morphologically, immunohistochemically and by TUNEL-method. Cells positive to caspase-3 had brown stained nuclei or nuclear fragmentations. At earliest stages, 6% of ganglion population were caspase-3 positive cells. Later on, a significant increase in number of caspase-3 positive cells appeared, particularly in the ventral part of ganglia (12%), and subsequently decreased to 6%. TUNEL-positive cells had the same distribution pattern as caspase-3 positive cells. Bax-positive cells followed the developmental pattern similar to caspase-3 cells, changing in range between 20% and 32%. There were 8% of bcl-2 positive cells at earliest stages. They increased significantly in dorsal part of the ganglion during the 7th week (28%), and than dropped to 15% by the end of the 8th week. These findings suggest a ventro-dorsal course of development in human spinal ganglia. Number of bcl-2, bax and caspase-3 positive cells changed in a temporally and spatially restricted manner, coincidently with ganglion differentiation. While apoptosis might control cell number, bcl-2 could act in suppression of apoptosis and enhancement of cell differentiation.

An overview of Bcl-2 expression in histopathological variants of basal cell carcinoma, squamous cell carcinoma, actinic keratosis and seborrheic keratosis.

The Bcl-2 protein has been shown to suppress cell death and protects cell against apoptosis induced by different death-inducing signals. In this study the authors have analyzed imunohistochemically the expression of Bcl-2 protein in the histopathological variants of the most common malignant tumors of the skin--basal cell carcinoma (BCC) and squamous cell tumor (SCC), as well as in the precancerous lesion actinic keratosis (AK) and in benign tumor seborrheic keratosis (SK). Bcl-2 expression in solid, adenoid and cystic variants of BCC exhibited immunoreactivity of tumor stroma with more intense staining among peripheral palisading cells. Morphoeic variant demonstrated reduced amount of Bcl-2 expression. Among SCC in all samples, tumor tissue lack to express Bcl-2 positivity. In cases of hypertrophic and atrophic variants of AK, Bcl-2 expression was confined to basal cell layer, as well as in one case of hypertrophic variant in suprabasal cells. In three histological variants of SK expresseion of Bcl-2 protein was in areas of basaloid proliferation, while in areas of squamous differentiation was negative. In clonal variant immunostaining was positive among cells in characteristic "nests" Distribution of Bcl-2 protein expression in solid, adenoid and cystic variant of BCC showed that peripheral proliferating cells are protected against apoptosis what permits tumor growth. In morpheaform variant reduced amount of Bcl-2 expression indicated that this variant of BCC has increased cell proliferation, and in practice shows tendency for recurrence and difficulties to eradicate. Bcl-2 expression supports the observation that tumor cells are derived from basal keratinocytes. In SCC, lack of Bcl-2 expression indicates that origin of tumor cells is from more differentiated suprabasal keratinocytes. In AK results suggest that immunoreactivity is regulated with respect of the keratinocyte's differentiation status, but not closely correlate with proliferative rate.

Role of mitotic, pro-apoptotic and anti-apoptotic factors in human kidney development.

The expression pattern of mitotic Ki-67 and anti-apoptotic bcl-2 proteins, as well as apoptotic caspase-3 and p53 proteins, were investigated in the human mesonephros and metanephros of 5-9 week-old human conceptuses. Apoptotic cells were additionally detected using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) method. Between the 5th and 7th developmental weeks Ki-67, caspase-3 and TUNEL-positive cells characterized all mesonephric structures, indicating importance of cell proliferation in the growth of the mesonephros and role of apoptosis in nephrogenesis. From the 7th week on, p53 and bcl-2 positive cells appeared in the mesonephros as well. Regressive changes in the mesonephros could be regulated by activation of p53, while bcl-2 could contribute to selective survival of some tubules giving rise to adult structures. In the early human metanephros (5-7 weeks), Ki-67 positive cells characterized all metanephric structures, indicating a role of cell proliferation in branching of the ureteric bud and in nephron formation. During the same period bcl-2, caspase-3 and TUNEL-positive cells were found only in the metanephric mesenchyme and nephrons. Bcl-2 protein probably protected nephrons from apoptosis, while caspase-3 protein controlled cell death in the mesenchyme. At later stages (7-9-weeks), appearance of p53-expressing cells could participate in further morphogenesis of the metanephric collecting system. The factors investigated had a spatially and temporally restricted pattern of appearance in developing kidneys. Changes in that pattern might lead to serious disturbances of kidney formation and function in early childhood.

Cell death in developing human spinal cord.

Cell death in the developing human spinal cord was investigated in 5-12 week human conceptuses using immunohistochemical and TUNEL methods. Expression of pro-apoptotic (Fas-receptor, caspase-3) and anti-apoptotic (bcl-2) markers and marker for internucleosomal fragmentation (TUNEL) were analysed in the cranial and caudal parts of the human spinal cord. In early developmental stages (5-6 weeks) of the cranial spinal cord, bcl-2 positive cells were seen in the ventricular zone and in the roof plate, while in the caudal part they were seen surrounding the central lumen. Subsequently, bcl-2 expression appeared in the basal plates of the grey matter and in the spinal ganglia, and from the seventh week on they also appeared in the intermediate horn of the grey matter. In the fetal period, bcl-2 expression appeared in the dorsal horns of the grey matter (9 weeks) but ceased in the ventricular zone (12 weeks) . In the trunk region, TUNEL-positive cells were found in ventricular and mantle zones along the whole length of the spinal cord. Caspase-3 positive cells and Fas-receptor positive cells appeared only in the grey matter of the cranial segments (head and trunk) of the spinal cord, but they were missing in the caudal parts. Caspase-3 dependant pathway, probably activated by Fas-receptor, seems to operate only in the cranial part of the human spinal cord. In the caudal (sacrococcygeal and tail) parts, cells seem to die by caspase-3 independent pathway. The interplay of pro-apoptotic and anti-apoptotic factors may be associated with cranial spinal cord morphogenesis, adjustment of cells number and selective survival of neurons, while in the caudal regions these factors cause massive cell death associated with regression of the caudal spinal cord.

Distinct membrane effects of spinal nerve ligation on injured and adjacent dorsal root ganglion neurons in rats.

BACKGROUND: Painful peripheral nerve injury results in disordered sensory neuron function that contributes to the pathogenesis of neuropathic pain. However, the relative roles of neurons with transected axons versus intact adjacent neurons have not been resolved. An essential first step is identification of electrophysiologic changes in these two neuronal populations after partial nerve damage. METHODS: Twenty days after spinal nerve ligation (SNL), intracellular recordings were obtained from axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent, intact L4 neurons, as well as from control neurons and others subjected to sham-SNL surgery. RESULTS: Pronounced electrophysiologic changes were seen only in L5 neurons after SNL. Both Aalpha/beta and Adelta neuron types showed increased action potential duration, decreased afterhyperpolarization amplitude and duration, and decreased current threshold for action potential initiation. Aalpha/beta neurons showed resting membrane potential depolarization, and increased repetitive firing during sustained depolarization developed in Adelta neurons. The afterhyperpolarization duration in neurons with C fibers shortened after axotomy. In contrast to the axotomized L5 neurons, neighboring L4 neurons showed no changes in action potential duration, afterhyperpolarization dimensions, or excitability after SNL. Depolarization rate (dV/dt) increased after SNL in L4 Aalpha/beta and Adelta neurons but decreased in L5 neurons. Time-dependent rectification during hyperpolarizing current injection (sag) was greater after SNL in Aalpha/beta L4 neurons compared with L5. Sham-SNL surgery produced only a decreased input resistance in Aalpha/beta neurons and a decreased conduction velocity in medium-sized cells. In the L5 ganglion after axotomy, a novel set of neurons, consisting of 24% of the myelinated population, exhibited long action potential durations despite myelinated neuron conduction velocities, particularly depolarized resting membrane potential, low depolarization rate, and absence of sag. CONCLUSIONS: These findings indicate that nerve injury-induced electrical instability is restricted to axotomized neurons and is absent in adjacent intact neurons.

Detection of neuropathic pain in a rat model of peripheral nerve injury.

BACKGROUND: Behavioral criteria that confirm neuropathic pain in animal injury models are undefined. Therefore, the authors sought clinically relevant measures that distinguish pain behavior of rats with peripheral nerve injury from those with sham injury. METHODS: The authors examined mechanical and thermal sensory sensitivity, comparing responses at baseline to responses after spinal nerve ligation (SNL group), sham nerve injury (sham group), or skin incision alone (control group). RESULTS: Substantial variance was evident in all sensory tests at baseline. After surgery, tests using brush, cold, or heat stimulation showed minimal distinctions between surgical groups. Postsurgical thresholds for flexion withdrawal from mechanical stimulation with von Frey fibers were decreased bilaterally in SNL and sham groups. In contrast, the probability of a complex hyperalgesia-type response with prolonged elevation, shaking, or licking of the paw was selectively increased on the ipsilateral side in the SNL group. Nonetheless, the effect of SNL on behavior was inconsistent, regardless of the sensory test. The behavioral measure that best distinguishes between SNL and sham groups and thereby best identifies animals with successful SNL-induced neuropathic pain is increased ipsilateral postsurgical probability of a hyperalgesia-type response to noxious mechanical stimulation. Using receiver operating characteristics analysis, mechanical hyperalgesia identifies a local SNL effect in approximately 60% of animals when specificity is required to be 90% or higher. CONCLUSIONS: Simple withdrawal from von Frey tactile stimulation, although frequently used, is not a valid measure of peripheral nerve injury pain in rats, whereas a complex hyperalgesic-type response is a specific neuropathy-induced behavior.