Vesna Jevtovic-Todorovic

Nerve injury reduced the γ-aminobutyric acid (GABA) receptor type A α₂subunit expression in dorsal root ganglia (DRG).In vivoknockdown of the α₂subunit in the DRG enhanced nociception whereas pharmacologic prevention of GABA uptake reduced pain behavior. The results are consistent with the notion that modulation of GABAergic tone in the DRG may offer a novel therapeutic option for the treatment of neuropathic pain.Background: Neuropathic pain (NPP) is likely the result of repetitive high-frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn. Drugs that promote γ-aminobutyric acid (GABA) activity in the dorsal horn provide partial relief of neuropathic symptoms. The authors examined howin vivosilencing of the GABA receptor type A (GABA_A) α₂gene in dorsal root ganglia (DRG) controls NPP.Methods: After crush injury to the right sciatic nerve of female rats, the α₂GABA_Aantisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG.In vivobehavioral assessment of nociception was conducted before the injury and ensuing 10 days (n = 4 to 10). In vitroquantification of α₂GABA_Aprotein and electrophysiological studies of GABA_Acurrents were performed on acutely dissociated L5 DRG neurons at relevant time points (n = 6 to 14).Results: NPP postcrush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α₂subunit expression in the ipsilateral DRG (approximately 30%). Selective down-regulation of α₂expression in DRGs significantly worsens mechanical (2.55 ± 0.75 to 5.16 ± 1.16) and thermal (7.97 ± 0.96 to 5.51 ± 0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33 ± 0.40 to 5.00 ± 0.33) and thermal (10.80 ± 0.29 to 7.34 ± 0.81) hypersensitivity in sham animals (data shown as mean ± SD). Conversely, up-regulation of endogenous GABAviablockade of its uptake in DRG alleviates NPP.Conclusion: The GABA_Areceptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represents promising target for novel pain therapies.

We recently showed that streptozotocin (STZ) injections in rats lead to the development of painful peripheral diabetic neuropathy (PDN) accompanied by enhancement of Ca_V3.2 T-type calcium currents (T-currents) and hyperexcitability in dorsal root ganglion (DRG) neurons. Here we used the classical peripherally acting T-channel blocker mibefradil to examine the role of Ca_V3.2 T-channels as pharmacological targets for treatment of painful PDN. When administered intraperitoneally (i.p.), at clinically relevant doses, mibefradil effectively alleviated heat, cold and mechanical hypersensitivities in STZ-treated diabetic rats in a dose-dependent manner. We also found that Ca_V3.2 antisense (AS)-treated diabetic rats exhibit a significant decrease in painful PDN compared with mismatch antisense (MIS)-treated diabetic rats. Co-treatment with mibefradil (9 mg/kg i.p.) resulted in reversal of heat, cold and mechanical hypersensitivity in MIS-treated but not in AS-treated diabetic rats, suggesting that mibefradil and Ca_V3.2 AS share the same cellular target. Using patch-clamp recordings from acutely dissociated DRG neurons, we demonstrated that mibefradil similarly blocked T-currents in diabetic and healthy rats in a voltage-dependent manner by stabilizing inactive states of T-channels. We conclude that antihyperalgesic and antiallodynic effects of mibefradil in PDN are at least partly mediated by inhibition of Ca_V3.2 channels in peripheral nociceptors. Hence, peripherally acting voltage-dependent T-channel blockers could be very useful in the treatment of painful symptoms of PDN.

Exposure to general anaesthesia during critical stages of brain development results in long-lasting cognitive impairment. Co-administration of protective agents could minimize the detrimental effects of anaesthesia. Co-administration of R(+)pramipexole (PPX), a synthetic aminobenzothiazol derivative that restores mitochondrial integrity, prevents anaesthesia-induced mitochondrial and neuronal damage and prevents early development of cognitive impairment. Here, we determine the protective effects of PPX into late adulthood in male and female rats. Postnatal day 7 rats of both sexes were exposed to mock anaesthesia or combined midazolam, nitrous oxide, and isoflurane anaesthesia for 6 h with or without PPX. Cognitive abilities were assessed between 5 and 7 months of age using Morris water maze spatial navigation tasks. Examination of spatial reference memory revealed that female, but not male, neonatal rats exposed to anaesthesia showed slowing of acquisition rates, which was significantly improved with PPX treatment. Examination of memory retention revealed that both male and female anaesthesia-treated rats have impaired memory retention performance compared with sham controls. Co-treatment with PPX resulted in improvement in memory retention in both sexes. PPX provides long-lasting protection against cognitive impairment known to occur when very young animals are exposed to anaesthesia during the peak of brain development. Anaesthesia-induced cognitive impairment appears to be sex-specific with females being more vulnerable than males, suggesting that they could benefit more from early prevention.

Background: General anesthetics induce apoptotic neurodegeneration in the developing mammalian brain. General anesthesia (GA) also causes significant disturbances in mitochondrial morphogenesis during intense synaptogenesis. Mitochondria are dynamic organelles that undergo remodeling via fusion and fission. The fine balance between these two opposing processes determines mitochondrial morphometric properties, allowing for their regeneration and enabling normal functioning. As mitochondria are exquisitely sensitive to anesthesia-induced damage, we examined how GA affects mitochondrial fusion/fission. Methods: Seven-day-old rat pups received anesthesia containing a sedative dose of midazolam followed by a combined nitrous oxide and isoflurane anesthesia for 6 h. Results: GA causes 30% upregulation of reactive oxygen species (n = 3–5 pups/group), accompanied by a 2-fold downregulation of an important scavenging enzyme, superoxide dismutase (n = 6 pups/group). Reactive oxygen species upregulation is associated with impaired mitochondrial fission/fusion balance, leading to excessive mitochondrial fission. The imbalance between fission and fusion is due to acute sequestration of the main fission protein, dynamin-related protein 1, from the cytoplasm to mitochondria, and its oligomerization on the outer mitochondrial membrane. These are necessary steps in the formation of the ring-like structures that are required for mitochondrial fission. The fission is further promoted by GA-induced 40% downregulation of cytosolic mitofusin-2, a protein necessary for maintaining the opposing process, mitochondrial fusion (n = 6 pups/group). Conclusions: Early exposure to GA causes acute reactive oxygen species upregulation and disturbs the fine balance between mitochondrial fission and fusion, leading to excessive fission and disturbed mitochondrial morphogenesis. These effects may play a causal role in GA-induced developmental neuroapoptosis.

Normal cognitive development depends on the timely formation of meaningful neuronal circuitries. These, in turn, depend on the proper formation and functioning of neuronal synapses, which control the flow of information between neurons. The time period when synapse formation is most intense is referred to as synaptogenesis, coinciding with the peak of brain development. The latest animal and human research suggests that general anesthetics, which act by modulating the fine balance in neurotransmission, may disturb the fine homeostasis necessary for neuronal signaling, resulting in morphometric and functional disturbances of developing synapses in synaptogenesis. Anesthesia-induced impairment of synaptogenesis is strongly age-dependant. At a younger stage, neurons respond by decreasing synaptic densities. But in later development, they respond by overly upregulating synapse formation. Although a direct causal link between disturbed synaptogenesis and behavioral development is not yet established, several animal studies have confirmed that cognitive development of rodents and non-human primates could be permanently impaired after a single exposure to clinically-relevant general anesthetics. Clinical evidence is now beginning to emerge suggesting that very young children may be susceptible to anesthesiainduced impairment of behavioral development, cognitive in particular. This review will summarize some of the presently available evidence regarding anesthesia-induced effects on developmental synaptogenesis and intellectual functioning.

Background: Clinically used general anesthetics, alone or in combination, are damaging to the developing mammalian brain. In addition to causing widespread apoptotic neurodegeneration in vulnerable brain regions, exposure to general anesthesia at the peak of synaptogenesis causes learning and memory deficiencies later in life. In vivo rodent studies have suggested that activation of the intrinsic (mitochondria-dependent) apoptotic pathway is the earliest warning sign of neuronal damage, suggesting that a disturbance in mitochondrial integrity and function could be the earliest triggering events. Methods: Because proper and timely mitochondrial morphogenesis is critical for brain development, the authors examined the long-term effects of a commonly used anesthesia combination (isoflurane, nitrous oxide, and midazolam) on the regional distribution, ultrastructural properties, and electron transport chain function of mitochondria, as well as synaptic neurotransmission, in the subiculum of rat pups. Results: This anesthesia, administered at the peak of synaptogenesis, causes protracted injury to mitochondria, including significant enlargement of mitochondria (more than 30%, P < 0.05), impairment of their structural integrity, an approximately 28% increase in their complex IV activity (P < 0.05), and a twofold decrease in their regional distribution in presynaptic neuronal profiles (P < 0.05), where their presence is important for the normal development and functioning of synapses. Consequently, the authors showed that impaired mitochondrial morphogenesis is accompanied by heightened autophagic activity, decrease in mitochondrial density (approximately 27%, P < 0.05), and long-lasting disturbances in inhibitory synaptic neurotransmission. The interrelation of these phenomena remains to be established. Conclusion: Developing mitochondria are exquisitely vulnerable to general anesthesia and may be important early target of anesthesia-induced developmental neurodegeneration.

Due to increased frequency of surgical interventions, infants and young children are exposed to anesthesia, often repeatedly, during an extremely delicate period of brain development. We review new evidence that continues to challenge the safety of this practice. In animal models, anesthesia impairs normal synapse development and sculpting, which are crucial elements of developmental synaptogenesis. This age-dependent phenomenon is caused in part by actin cytoskeleton disorganization and impaired dendritic branching. Recent evidence also suggests that developing glia are sensitive to anesthesia-induced toxicity, which is manifested as stunted growth, delayed maturation, and disturbed process formation. Newly published findings in nonhuman primates, which report long-lasting cognitive impairment, stress the potential seriousness of anesthesia-induced developmental neurotoxicity. Although clinical importance remains to be substantiated, results to date do indicate that exposure of animals to general anesthesia during active synaptogenesis is most detrimental. Accordingly, it is essential to determine when synaptogenesis begins and ends in developing humans. It is also imperative that effective preventive techniques be developed so that existing anesthetics can be used with minimum risk of neurotoxic side-effects of anesthesia.