Standards for preclinical research and publications in developmental anaesthetic neurotoxicity: expert opinion statement from the SmartTots preclinical working group.

In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work.

Novel neuroactive steroid with hypnotic and T-type calcium channel blocking properties exerts effective analgesia in a rodent model of post-surgical pain.

BACKGROUND AND PURPOSE: Neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) is a novel hypnotic and voltage-dependent blocker of T-type calcium channels. Here, we examine its potential analgesic effects and adjuvant anaesthetic properties using a post-surgical pain model in rodents. EXPERIMENTAL APPROACH: Analgesic properties of 3ß-OH were investigated in thermal and mechanical nociceptive tests in sham or surgically incised rats and mice, with drug injected either systemically (intraperitoneal) or locally via intrathecal or intraplantar routes. Hypnotic properties of 3ß-OH and its use as an adjuvant anaesthetic in combination with isoflurane were investigated using behavioural experiments and in vivo EEG recordings in adolescent rats. KEY RESULTS: A combination of 1% isoflurane with 3ß-OH (60 mg·kg-1 , i.p.) induced suppression of cortical EEG and stronger thermal and mechanical anti-hyperalgesia during 3 days post-surgery, when compared to isoflurane alone and isoflurane with morphine. 3ß-OH exerted prominent enantioselective thermal and mechanical antinociception in healthy rats and reduced T-channel-dependent excitability of primary sensory neurons. Intrathecal injection of 3ß-OH alleviated mechanical hyperalgesia, while repeated intraplantar application alleviated both thermal and mechanical hyperalgesia in the rats after incision. Using mouse genetics, we found that CaV 3.2 T-calcium channels are important for anti-hyperalgesic effect of 3ß-OH and are contributing to its hypnotic effect. CONCLUSION AND IMPLICATIONS: Our study identifies 3ß-OH as a novel analgesic for surgical procedures. 3ß-OH can be used to reduce T-channel-dependent excitability of peripheral sensory neurons as an adjuvant for induction and maintenance of general anaesthesia while improving analgesia and lowering the amount of volatile anaesthetic needed for surgery.

Hyperexcitability of rat thalamocortical networks after exposure to general anesthesia during brain development.

Prevailing literature supports the idea that common general anesthetics (GAs) cause long-term cognitive changes and neurodegeneration in the developing mammalian brain, especially in the thalamus. However, the possible role of GAs in modifying ion channels that control neuronal excitability has not been taken into consideration. Here we show that rats exposed to GAs at postnatal day 7 display a lasting reduction in inhibitory synaptic transmission, an increase in excitatory synaptic transmission, and concomitant increase in the amplitude of T-type calcium currents (T-currents) in neurons of the nucleus reticularis thalami (nRT). Collectively, this plasticity of ionic currents leads to increased action potential firing in vitro and increased strength of pharmacologically induced spike and wave discharges in vivo. Selective blockade of T-currents reversed neuronal hyperexcitability in vitro and in vivo. We conclude that drugs that regulate thalamic excitability may improve the safety of GAs used during early brain development.

Propofol-induced changes in neurotrophic signaling in the developing nervous system in vivo.

Several studies have revealed a role for neurotrophins in anesthesia-induced neurotoxicity in the developing brain. In this study we monitored the spatial and temporal expression of neurotrophic signaling molecules in the brain of 14-day-old (PND14) Wistar rats after the application of a single propofol dose (25 mg/kg i.p). The structures of interest were the cortex and thalamus as the primary areas of anesthetic actions. Changes of the protein levels of the brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), their activated receptors tropomyosin-related kinase (TrkA and TrkB) and downstream kinases Akt and the extracellular signal regulated kinase (ERK) were assessed by Western immunoblot analysis at different time points during the first 24 h after the treatment, as well as the expression of cleaved caspase-3 fragment. Fluoro-Jade B staining was used to follow the appearance of degenerating neurons. The obtained results show that the treatment caused marked alterations in levels of the examined neurotrophins, their receptors and downstream effector kinases. However, these changes were not associated with increased neurodegeneration in either the cortex or the thalamus. These results indicate that in the brain of PND14 rats, the interaction between Akt/ERK signaling might be one of important part of endogenous defense mechanisms, which the developing brain utilizes to protect itself from potential anesthesia-induced damage. Elucidation of the underlying molecular mechanisms will improve our understanding of the age-dependent component of anesthesia-induced neurotoxicity.

T-type voltage-gated calcium channels as targets for the development of novel pain therapies.

It is well recognized that voltage-gated calcium (Ca(2+)) channels modulate the function of peripheral and central pain pathways by influencing fast synaptic transmission and neuronal excitability. In the past, attention focused on the modulation of different subtypes of high-voltage-activated-type Ca(2+) channels; more recently, the function of low-voltage-activated or transient (T)-type Ca(2+) channels (T-channels) in nociception has been well documented. Currently, available pain therapies remain insufficient for certain forms of pain associated with chronic disorders (e.g. neuropathic pain) and often have serious side effects. Hence, the identification of selective and potent inhibitors and modulators of neuronal T-channels may help greatly in the development of safer, more effective pain therapies. Here, we summarize the available information implicating peripheral and central T-channels in nociception. We also discuss possible future developments aimed at selective modulation of function of these channels, which are highly expressed in nociceptors.

Are neuroactive steroids promising therapeutic agents in the management of acute and chronic pain?

Neuroactive steroids with potentiating effects on GABA(A) channels and inhibitory effects on T-type Ca2+ channels which are located in peripheral sensory neurons are potent modulators of pain perception. The focus of this review is on peripheral anti-nociceptive properties of 5alpha- and 5beta-reduced neuroactive steroids with either selective or combined modulatory action on GABA(A) and T-type Ca2+ channel-mediated neurotransmission. We report that these neuroactive steroids are very effective in alleviating peripheral nociception in both acute and chronic pain conditions in animal models of pain. We believe that promising animal data warrant the exploration of their usefulness in clinical settings especially considering the fact that chronic pain sufferers are often young and otherwise healthy people.

General anesthesia activates BDNF-dependent neuroapoptosis in the developing rat brain.

Brain-derived neurotrophic factor (BDNF) is important in supporting neuronal development. BDNF imbalance due to excessive neuronal inhibition can result in the apoptotic degeneration of developing neurons. Since general anesthetics cause profound depression of neuronal activity and are known to induce widespread degeneration in the developing brain, we studied their potential to activate BDNF-mediated developmental neuroapoptosis. When P7 rats (at the peak of brain development) were exposed to a commonly-used and highly pro-apoptotic anesthesia protocol (midazolam, isoflurane, nitrous oxide) for a period of 2, 4 or 6 h, we found that anesthesia modulates the key steps in BDNF-activated apoptotic cascade in two of the most vulnerable brain regions--cerebral cortex and thalamus in time-dependent fashion by activating both Trk-dependent (in thalamus) and Trk-independent p75NTR dependent (in cerebral cortex) neurotrophic pathways. beta-estradiol, a sex hormone that upregulates the protein levels of the activated Akt, protects against anesthesia-induced neuroapoptosis.

CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus.

Although T-type Ca(2+) channels in the thalamus play a crucial role in determining neuronal excitability and are involved in sensory processing and pathophysiology of epilepsy, little is known about the molecular mechanisms involved in their regulation. Here, we report that reducing agents, including endogenous sulfur-containing amino acid l-cysteine, selectively enhance native T-type currents in reticular thalamic (nRT) neurons and recombinant Ca(V)3.2 (alpha1H) currents, but not native and recombinant Ca(V)3.1 (alpha1G)- and Ca(V)3.3 (alpha1I)-based currents. Consistent with this data, T-type currents of nRT neurons from transgenic mice lacking Ca(V)3.2 channel expression were not modulated by reducing agents. In contrast, oxidizing agents inhibited all native and recombinant T-type currents non-selectively. Thus, our findings directly demonstrate that Ca(V)3.2 channels are the main molecular substrate for redox regulation of neuronal T-type channels. In addition, because thalamic T-type channels generate low-threshold Ca(2+) spikes that directly correlate with burst firing in these neurons, differential redox regulation of these channels may have an important function in controlling cellular excitability in physiological and pathological conditions and fine-tuning of the flow of sensory information into the central nervous system.