Discordance of Gross and Histologic Findings in Estimating the Range of Fire of Gunshot Wounds.

Forensic pathologist use soot and/or stippling surrounding entrance gunshot wounds in categorizing range of fire. If absent, some pathologists suggest utilizing histology when the range is diagnostically critical. This study investigates the concordance of macroscopic and microscopic findings in estimating range of fire by evaluating gunshot entrance and exit wounds made through human tissue analogs at defined distances using two handgun calibers. Examination of over 150 entrance wounds verified the ease of visually detecting soot from a muzzle distance of contact to 1 foot (30.5 cm), and its absence at 9 feet (274.3 cm). Distinctly apparent was bullet wipe surrounding the entrance wounds regardless of muzzle distance. Although variations existed, dark material was histologically identified in many skin, soft tissue, and bone sections at all ranges with both calibers. These nonparallel results decrease the dependability of histology for range of fire estimation and reinforce using gross observation.

Effect of electrical field stimulation on dorsal root ganglion neuronal function.

OBJECTIVES: Neural stimulation may provide analgesia for a variety of painful conditions. Activation of primary sensory neurons, which underlies pain relief by spinal cord stimulation, also may be achieved by stimulation at the level of the dorsal root ganglion (DRG). The DRG also is a site of pain pathogenesis, particularly in neuropathic pain. We therefore examined the hypothesis that field stimulation of the DRG directly suppresses excitability of sensory neurons. MATERIALS AND METHODS: Intercellular Ca2+ level (Fura-2 microfluorimetry) and membrane potential were recorded in excised rat DRGs with ganglionic field stimulation (GFS) delivered by wire electrodes in the bath solution adjacent to the DRG. Neuronal excitability was evaluated by number of action potentials (APs) generated during neuronal depolarization, conduction velocity during axonal stimulation, and AP propagation failure. These were measured before and after 90 sec of GFS at 60 Hz. Data analysis employed chi-square, paired t-test, and analysis of variance. RESULTS: GFS using 400-µsec pulses and 30 V generated Ca2+ influx, indicative of DRG neuronal activation. Fewer neurons were able to fire one or more APs after GFS (N = 23) than in control neurons without GFS (N = 24, p < 0.05), and fewer neurons were able to generate multiple APs after GFS compared with time controls (p < 0.05). GFS significantly reduced conduction velocity compared with baseline before GFS (N = 16, p < 0.05) while there was no change in the controls (N = 18). The peak rate at which APs could be propagated was reduced in 9 of 16 neurons by GFS, but propagation efficiency was reduced in only 4 of 18 control neurons (p < 0.05), and the total number of APs generated in an ensemble of stimuli at different frequencies was reduced by GFS (N = 16, p < 0.05) but not in time controls (N = 18). CONCLUSIONS: Our findings indicate that direct excitation of the DRG by electrical fields reduces neuronal excitability and may provide a new analgesic approach.

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.

Ca²âº-dependent regulation of Ca²âº currents in rat primary afferent neurons: role of CaMKII and the effect of injury.

Currents through voltage-gated Ca²âº channels (I(Ca)) may be regulated by cytoplasmic Ca²âº levels ([Ca²âº](c)), producing Ca²âº-dependent inactivation (CDI) or facilitation (CDF). Since I(Ca) regulates sensory neuron excitability, altered CDI or CDF could contribute to pain generation after peripheral nerve injury. We explored this by manipulating [Ca²âº](c) while recording I(Ca) in rat sensory neurons. In uninjured neurons, elevating [Ca²âº](c) with a conditioning prepulse (-15 mV, 2 s) inactivated I(Ca) measured during subsequent test pulses (-15 mV, 5 ms). This inactivation was Ca²âº-dependent (CDI), since it was decreased with elimination of Ca²âº influx by depolarization to above the I(Ca) reversal potential, with high intracellular Ca²âº buffering (EGTA 10 mm or BAPTA 20 mm), and with substitution of Ba²âº for extracellular Ca²âº, revealing a residual voltage-dependent inactivation. At longer latencies after conditioning (>6 s), I(Ca) recovered beyond baseline. This facilitation also proved to be Ca²âº-dependent (CDF) using the protocols limiting cytoplasmic Ca²âº elevation. Ca²âº/calmodulin-dependent protein kinase II (CaMKII) blockers applied by bath (KN-93, myristoyl-AIP) or expressed selectively in the sensory neurons (AIP) reduced CDF, unlike their inactive analogues. Protein kinase C inhibition (chelerythrine) had no effect. Selective blockade of N-type Ca²âº channels eliminated CDF, whereas L-type channel blockade had no effect. Following nerve injury, CDI was unaffected, but CDF was eliminated in axotomized neurons. Excitability of sensory neurons in intact ganglia from control animals was diminished after a similar conditioning pulse, but this regulation was eliminated by injury. These findings indicate that I(Ca) in sensory neurons is subject to both CDI and CDF, and that hyperexcitability following injury-induced loss of CDF may result from diminished CaMKII activity.

Store-operated Ca2+ entry in sensory neurons: functional role and the effect of painful nerve injury.

Painful nerve injury disrupts levels of cytoplasmic and stored Ca(2+) in sensory neurons. Since influx of Ca(2+) may occur through store-operated Ca(2+) entry (SOCE) as well as voltage- and ligand-activated pathways, we sought confirmation of SOCE in sensory neurons from adult rats and examined whether dysfunction of SOCE is a possible pathogenic mechanism. Dorsal root ganglion neurons displayed a fall in resting cytoplasmic Ca(2+) concentration when bath Ca(2+) was withdrawn, and a subsequent elevation of cytoplasmic Ca(2+) concentration (40 ± 5 nm) when Ca(2+) was reintroduced, which was amplified by store depletion with thapsigargin (1 µm), and was significantly reduced by blockers of SOCE, but was unaffected by antagonists of voltage-gated membrane Ca(2+) channels. We identified the underlying inwardly rectifying Ca(2+)-dependent I(CRAC) (Ca(2+) release activated current), as well as a large thapsigargin-sensitive inward current activated by withdrawal of bath divalent cations, representing SOCE. Molecular components of SOCE, specifically STIM1 and Orai1, were confirmed in sensory neurons at both the transcript and protein levels. Axonal injury by spinal nerve ligation (SNL) elevated SOCE and I(CRAC). However, SOCE was comparable in injured and control neurons when stores were maximally depleted by thapsigargin, and STIM1 and Orai1 levels were not altered by SNL, showing that upregulation of SOCE after SNL is driven by store depletion. Blockade of SOCE increased neuronal excitability in control and injured neurons, whereas injured neurons showed particular dependence on SOCE for maintaining levels of cytoplasmic and stored Ca(2+), which indicates a compensatory role for SOCE after injury.

Calcium signaling in intact dorsal root ganglia: new observations and the effect of injury.

BACKGROUND: Ca is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca recording techniques use high K or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. METHODS: Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. RESULTS: Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Abeta-type neurons. Unitary Ca transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca transients in C-type neurons, but increased transients in Abeta-type neurons. CONCLUSIONS: Refined observation of Ca signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state.