Damir Sapunar

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.

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 Aα/β and A&dgr; neuron types showed increased action potential duration, decreased afterhyperpolarization amplitude and duration, and decreased current threshold for action potential initiation. Aα/β neurons showed resting membrane potential depolarization, and increased repetitive firing during sustained depolarization developed in A&dgr; 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 Aα/β and A&dgr; neurons but decreased in L5 neurons. Time-dependent rectification during hyperpolarizing current injection (sag) was greater after SNL in Aα/β L4 neurons compared with L5. Sham-SNL surgery produced only a decreased input resistance in Aα/β 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.

Dorsal root ganglion - a potential new therapeutic target for neuropathic pain.

A regional approach can protect our patients from often unacceptable adverse effects produced by systematically applied drugs. Regional therapeutic approaches, as well as interventions at the level of the peripheral nervous system and particularly the dorsal root ganglion (DRG), represent an alternative to the systemic application of therapeutic agents. This article provides an overview of DRG anatomical peculiarities, explains why the DRG is an important therapeutic target, and how animal models of targeted drug delivery can help us in the translation of basic research into clinical practice.

Painful Peripheral Nerve Injury Decreases Calcium Current in Axotomized Sensory Neurons

Background:Reports of Ca2+ current (ICa) loss after injury to peripheral sensory neurons do not discriminate between axotomized and spared neurons. The spinal nerve ligation model separates axotomized from spared neurons innervating the same site. The authors hypothesized that ICa loss is a result of neuronal injury, so they compared axotomized L5 dorsal root ganglion neurons to spared L4 neurons, as well as neurons from rats undergoing skin incision alone. Methods:After behavioral testing, dissociated neurons from L4 and L5 dorsal root ganglia were studied in both current and voltage patch clamp modes. The biophysical consequence of ICa loss on the action potential was confirmed using selective ICa antagonists. Data were grouped into small, medium, and large cells for comparison. Results:Reduced ICa was predominantly a consequence of axotomy (L5 after spinal nerve ligation) and was most evident in small and medium neurons. ICa losses were associated with action potential prolongation in small and medium cells, whereas the amplitude and duration of after hyperpolarization was reduced in medium and large neurons. Blockade with Ca2+ channel antagonists showed that action potential prolongation and after hyperpolarization diminution were alike, attributable to the loss of ICa. Conclusion:Axotomy is required for ICa loss. ICa loss correlated with changes in the biophysical properties of sensory neuron membranes during action potential generation, which were due to ICa loss leading to decreased outward Ca2+-sensitive K+ currents. Taken together, these results suggest that neuropathic pain may be mediated, in part, by loss of ICa and the cellular processes dependent on Ca2+.

Modulators of Calcium Influx Regulate Membrane Excitability in Rat Dorsal Root Ganglion Neurons

BACKGROUND: Chronic neuropathic pain resulting from neuronal damage remains difficult to treat, in part, because of incomplete understanding of underlying cellular mechanisms. We have previously shown that inward Ca2+ flux (ICa) across the sensory neuron plasmalemma is decreased in a rodent model of chronic neuropathic pain, but the direct consequence of this loss of ICa on function of the sensory neuron has not been defined. We therefore examined the extent to which altered membrane properties after nerve injury, especially increased excitability that may contribute to chronic pain, are attributable to diminished Ca2+ entry. METHODS: Intracellular microelectrode measurements were obtained from A-type neurons of dorsal root ganglia excised from uninjured rats. Recording conditions were varied to suppress or promote ICa while biophysical variables and excitability were determined. RESULTS: Both lowered external bath Ca2+ concentration and blockade of ICa with bath cadmium diminished the duration and area of the after-hyperpolarization (AHP), accompanied by decreased current threshold for action potential (AP) initiation and increased repetitive firing during sustained depolarization. Reciprocally, elevated bath Ca2+ increased the AHP and suppressed repetitive firing. Voltage sag during neuronal hyperpolarization, indicative of the cation-nonselective H-current, diminished with decreased bath Ca2+, cadmium application, or chelation of intracellular Ca2+. Additional recordings with selective blockers of ICa subtypes showed that N-, P/Q, L-, and R-type currents each contribute to generation of the AHP and that blockade of any of these, and the T-type current, slows the AP upstroke, prolongs the AP duration, and (except for L-type current) decreases the current threshold for AP initiation. CONCLUSIONS: Taken together, our findings show that suppression of ICa decreases the AHP, reduces the hyperpolarization-induced voltage sag, and increases excitability in sensory neurons, replicating changes that follow peripheral nerve trauma. This suggests that the loss of ICa previously demonstrated in injured sensory neurons contributes to their dysfunction and hyperexcitability, and may lead to neuropathic pain.

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.

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

The peripheral terminals of sensory neurons encode physical and chemical signals into trains of action potentials (APs) and transmit these trains to the CNS. Although modulation of this process is thought to predominantly reside at synapses, there are also indications that AP trains are incompletely propagated past points at which axons branch. One such site is the T‐junction, where the single sensory neuron axon branches into peripheral and central processes. In recordings from sensory neurons of dorsal root ganglia excised from adult rats, we identified use‐dependent failure of AP propagation between the peripheral and central processes that results in filtering of rapid AP trains, especially in C‐type neurons. Propagation failure was regulated by membrane input resistance and Ca2+‐sensitive K+ and Cl− currents. Following peripheral nerve injury, T‐junction filtering is reduced in C‐type neurons, which may possibly contribute to pain generation.

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.

Extracting data from figures with software was faster, with higher interrater reliability than manual extraction

OBJECTIVES To compare speed and accuracy of graphical data extraction using manual estimation and open source software. STUDY DESIGN AND SETTING Data points from eligible graphs/figures published in randomized controlled trials (RCTs) from 2009 to 2014 were extracted by two authors independently, both by manual estimation and with the Plot Digitizer, open source software. Corresponding authors of each RCT were contacted up to four times via e-mail to obtain exact numbers that were used to create graphs. Accuracy of each method was compared against the source data from which the original graphs were produced. RESULTS Software data extraction was significantly faster, reducing time for extraction for 47%. Percent agreement between the two raters was 51% for manual and 53.5% for software data extraction. Percent agreement between the raters and original data was 66% vs. 75% for the first rater and 69% vs. 73% for the second rater, for manual and software extraction, respectively. CONCLUSIONS Data extraction from figures should be conducted using software, whereas manual estimation should be avoided. Using software for data extraction of data presented only in figures is faster and enables higher interrater reliability.

Depression of Ca2+/calmodulin‐dependent protein kinase II in dorsal root ganglion neurons after spinal nerve ligation

The enzyme calcium/calmodulin‐dependent protein kinase II (CaMKII) is associated with memory and its α isoform is critical for development of activity‐induced synaptic changes. Therefore, we hypothesized that CaMKII is involved in altered function of dorsal root ganglion (DRG) neurons after neuronal injury. To test this hypothesis, Sprague–Dawley rats were made hyperalgesic by L5 and L6 spinal nerve ligation (SNL), and changes in total phosphorylated and unphosphorylated CaMKII (tCaMKII) and phosphorylated form of its α isoform (pCaMKIIα) were analyzed using immunochemistry in different subpopulations of DRG. SNL did not induce any changes in tCaMKII between experimental groups, while the overall percentage of pCaMKIIα‐positive neurons in injured L5 DRG SNL (24.8%) decreased significantly when compared to control (41.7%). SNL did not change the percentage of pCaMKIIα/N52 colabeled neurons but decreased the percentage of N52‐negative nonmyelinated neurons that expressed pCaMKIIα from 27% in control animals to 11% after axotomy. We also observed a significant decrease in the percentage of small nonpeptidergic neurons labeled with IB4 (37.6% in control vs. 4.0% in L5 SNL DRG), as well as a decrease in the percentage of pCaMKIIα/IB4 colabeled neurons in injured L5 DRGs (27% in control vs. 1% in L5 DRG of SNL group). Our results show that reduction in pCaMKIIα levels following peripheral injury is due to the loss of IB4‐positive neurons. These results indicate that diminished afferent activity after axotomy may lead to decreased phosphorylation of CaMKIIα. J. Comp. Neurol. 518:64–74, 2010.