Neuronal apoptosis of the developing brain: Influence of anesthetics

Padmaja Durga, M.D., P.D.C.C. (Cardiac &Neuroanesthesia), Professor Department of Anesthesiology and Critical Care, Nizam’s Institute of Medical Sciences, Hyderabad.
Children who require surgical interventions, are exposed to many stressors including mental, pain, inflammatory and anesthesia, which could affect brain and behavioural development. There is an increasing concern regarding the risk of anaesthetic neurotoxicity in children. Compelling evidence has shown that exposure to all commonly used anesthetics and sedatives, with the possible exception of α2-adrenergic agonists can cause neurodegeneration in the developing brain, but the basis of this is not clear. Neurotoxicity induced by exposure to anesthestics in early life involves neuroapoptosis and impairment of neurodevelopmental processes such as neurogenesis, synaptogenesis and immature glial development. These effects may subsequently contribute to behaviour abnormalities in later life.
Neurobehavior Mechanisms
Rapid brain development affects cognitive, social and emotional growth during the first three years of a child’s life. Experimental studies have shown that learning and memory are impaired in animals exposed to general anesthesia at early life, based on results from neurocognitive tests. Most of these studies attribute this cognitive dysfunction to anesthetic-induced neuroapoptosis and impaired neurogenesis. However, human neurobehavior is undoubtedly complex, such that subtle impairments in neurobehavior resulting from anesthesia are not easily detected through current neuropsychological and neurobehavior tests. Evidence has also indicated other sources of stress (surgical trauma, pain etc.) during the perioperative period can increase the risk of stress-related neurocognitive problems well into the adult years[1] . It is difficult to evaluate the relationship between anesthesia and neurotoxicity in humans. It has been shown that anesthetic-induced neurotoxic effects can be compensated during growth and differentiation of the nervous system, and that neurobehavior disorders can be restored in later life[2] . The mechanisms of neurobehavioral abnormalities induced by anesthetic exposure in early life need further investigation as the findings will have a profound implication on clinical practice.
Physiological Basis for enhanced vulnerability of Developing Brain Vulnerable to anaesthetic Neuronal Toxicity
Perinatal life and early childhood are the most intensive periods of brain development, during which the fetus, infants and children undergo an eruption in neuronal proliferation, differentiation, synaptogenesis, and rapid development of dendrites to establish the complicated networks of the central nervous system.  The numbers of synapses and neurons halves during development. The loss of neurons occurs via apoptosis. General anesthetics not only induce neuroapoptosis but also affect neurodevelopmental processes at the peak of synaptogenesis via certain cellular mechanisms. Disruption of physiological processes may result in neurodevelopmental disorders. Evidence is mounting that anesthetic exposure leads to a number of molecular, cellular and behavioral changes
in the developing brain, and these effects can be harmful and long-lasting.[3]
Molecular Mechanisms of Neuronal toxicity in Developing Brain

Neuroapoptosis
Apoptosis, or cell suicide, is an organized energy-consuming process by which ‘‘unwanted’’ cells are removed from the organ. Apoptosis involves chromatin aggregation, the condensation of cellular organelles, and the development of apoptotic bodies that are readily consumed by phagocytosis. Apoptosis may be triggered via two pathways: intrinsic and extrinsic. Both pathways, however, share a final common sequence that identifies an important marker of apoptosis:  activation of the enzyme caspase 3.[4] Many factors can trigger apoptosis including normal growth and development, diseases, and anesthetics. Although the underlying molecular mechanisms of anesthetic neurotoxicity are not completely understood, mitochondrial dysfunction, altered calcium homeostasis and apoptosis-related proteins have been implicated.[5]
NMDA Receptors and GABAA Receptors
The GABA and NMDA receptors are indirectly involved in the balance of activity and, thus, the generation of trophic factors that drive cell proliferation, migration, differentiation, growth, dendritic maturation and apoptosis.[6] Anaesthetics act on GABA and NMDA receptors.
The toxic effects of NMDA receptor antagonists on the immature brain have been extensively explored. Ketamine, a NMDA receptor antagonist, is commonly used for pediatric anesthesia and analgesia. Although clinical evidence for ketamine neurotoxicity in children undergoing is inconclusive [7-10] , recent experimental studies have reported that ketamine causes neuronal cell death in developing animals[11, 12] . Ketamine may cause a compensatory up-regulation of NMDA receptors, subsequently triggering expression of apoptosis-related genes in the developing neurons.
Neuroscientists have realized gamma-aminobutyric acid type A (GABAA) receptor agonists can affect neurodevelopment. Currently, several anesthetics, sedatives or anticonvulsants used clinically acting on GABA A receptor agonists. They could suppress postnatal neurogenesis trigger widespread apoptotic cell death in developing rodent brain, eventually resulting in long-term neurobehavioral impairment.[13, 14]
Mitochondrial Perturbations
Mitochondria  also play essential roles in controlling apoptosis. Injured mitochondria could be a significant source of reactive oxygen species (ROS) which, if not scavenged properly, may cause excessive lipid peroxidation and damage of cellular membranes. Anesthesia can impair mitochondrial morphogenesis, integrity and function at the peak of synaptogenesis. This mitochondrial impairment may be central in anesthetic-induced acute neuroapoptosis and cognitive abnormalities in later life.  Anesthetics induces neurotoxicity through opening of the mitochondrial permeability transition pore (mPTP), elevation in ROS levels, reduction in mitochondrial membrane potential and adenosine-5'-triphosphate (ATP) production, and activation of caspase-3.[15-18] Peri-anesthesia treatment with an ROS scavenger or mitochondria protectant prevented anesthesia-induced cognitive impairment.
Dysregulation of Intracellular Ca2+ Homeostasis
Loss of loss of calcium regulation promotes several events including mitochondrial dysfunction and cytochrome C release, increased active caspase-3, growth cone collapse, as well as reduced neurite length and complexity. Downstream of calcium dysregulation are changes in expression of proteins related to the cytoskeleton, synapse, production of neurotransmitters, or calcium buffering. Volatile anesthetics including isoflurane and sevoflurane could induce intracellular calcium overload, which increases ROS and NO levels that could result in neuroapoptosis. Isoflurane can enhance the GABA-induced [Ca2+] increase and potentiate GABA A receptor-mediated synaptic voltage-dependent calcium channels (VDCCs).[13] Calcium oscillations increase CaMK II levels which would then promote neuronal synaptic plasticity, and synapsin levels.  A persistent intracellular Ca2+ concentration interferes with Ca2+ oscillation, which would affect neuronal synaptogenesis and also leads to neuronal apoptosis.[19]
Neuroinflammatory Pathway
Recent findings suggest that neuroinflammatory mediators such as cytokines may be involved in a number of key steps in the pathological cascade of events leading to anesthetic-induced neuronal injury[20, 21] . Nociceptive stimulation (e.g. surgical incision) with prolonged anesthesia exposure produces significantly more apoptosis than prolonged anesthesia alone in neonates during the synaptogenic period[22] . Both anesthesia and surgery can induce cytokines release in the central nervous system, leading to deleterious neurodevelopmental effects.
The BDNF Pathway
Neurotrophins such as brain-derived  neurotrophic factor (BDNF)  are chemicals of central importance in the regulation of the survival, differentiation, and maintenance of functions as synaptic plasticity in the developing brain. Evidence has shown that general anesthetics induce neuroapoptotic damage in the developing brain, at least in part via the –BDNF modulated apoptotic cascade. [23, 24]
Other Cellular Processes in Neurodevelopment
Neurogenesis
Anesthetics can cause the inhibition on maturation and proliferation of neuronal progenitor cells,[25, 26] , decrease the  pool of neural stem cells and decrease their self-renewal capacity.[27] The inflammatory cytokines induced by general anesthetics may also impair neural progenitor cells proliferation and alter their differentiation.[28] These changes could adversely result in late cognitive dysfunction after general anesthesia in age dependent manner.[29]

Dendritic Development
The dendritic spines are the postsynaptic sites of most excitatory axodendritic synapses in the brain. Genesis of dendritic filopodia and spines formation play a critical role in synaptogenesis. Impairment of synaptogenesis potentially interferes with the development of neural networks. Recent studies from fixed brain preparations have shown that exposure to ketamine[30] and isoflurane decreases synapse or spine density in hippocampus of neonatal rodents. On the other hand, exposure to anesthetics midazolam, propofol, or ketamine causes a significant increase in the density of dendritic spines.[31] The effects of anesthetic exposure on synaptic connectivity in the brain may depend on developmental stage level[32] and the dose of anesthetics. [33] The mechanisms underlying the effects on anesthetics on synaptogenesis remain unclear, but at least may, in part, involve blockade of NMDA receptor activity or potentiation of GABAA receptor activity.
Neurite Outgrowth
The effects of anesthetics on neurite outgrowth are reversible and transient.[34, 35] This makes anesthetics unlikely to induce cognitive dysfunction by this mechanism.
Glial Development
Astrocytes, the most abundant glial cells in brain, are necessary for the formation, function, stability and plasticity of synapses. Anesthetics may interfere at multiple levels to impair proper cytoskeletal development early, thereby disturbing glial growth and maturation.[36] The susceptibility of glial cells to anesthetic toxicity is age-dependent as well. The lethal anesthetic dose for immature glial cells and neural stem cells is greater than that for developing neurons.
Evidence from Animal Studies for neuronal toxicity of anesthetics
Studies demonstrated accelerated neuronal apoptosis in newborn rodents after they were exposed only to general anaesthetics such as ketamine[11] , propofol[37], nitrous oxide, sevoflurane, isoflurane[38] , desflurane, and benzodiazepines, without any other insults. In rats, the effect of anesthetics on apoptosis was greatest in 5–7 day olds. . Furthermore, more prolonged exposure to anesthetics as well as exposure to multiple anaesthetic agents in combination exacerbated the severity of the apoptosis. These findings cannot be translated to pediatric and obstetrics anesthesia practice for now but have potential implications.

Factors Influencing Neuronaltoxicity of Anesthetics
Anesthetic Exposure and Timing
Exposure Concentration and Duration
Even lower volatile anesthetic concentration (for example, 0.5 minimum alveolar concentration (MAC)) is usually used to protect against ischemic brain injury, can result in significant cytotoxicity. Subanesthetic concentration of halothane, sevoflurane and desflurane (0.1%, 0.3%, and 0.6% in 3 L/min O2, respectively) impair behavioral functions.[39]
Alterations in learning and memory functions are greater with desflurane than with halothane and sevoflurane. [40] Desflurane but not isoflurane treatment induces almost no apoptosis or neurocognitive dysfunction[41] Difference between the two volatile anesthetics may be due to a difference in the effects of these anesthetics on mitochondrial function. [42, 17] exposure to a combination of anesthetics (for example, nitrous oxide and desflurane) may cause more severe neuroapoptosis than to a single agent by itself.
Age Dependency of Apoptotic Neurodegeneration
Developing brain more susceptible to anesthetic-induced neurotoxicity compared to the mature brain. [43] Anesthetic effects on the brain during its growth spurt period have led us to recognize that a developmental insult can initiate a cascade of alterations in neurodevelopment which can be detected structurally or functionally.  Anesthetics are more likely to cause adverse effects if the exposure interferes with the cascade of  neurodevelopmental processes as during general anesthesia in pediatric or obstetric surgery.[44-47]
Since, every anaesthetic agent has the potential to induce apoptosis in neonatal neurones, it is important to consider whether the anaesthetic state itself promotes apoptosis in the neonatal period. It has been proposed that anaesthetic suppression of spontaneous neuronal activity might lead to insufficient neurotrophic factor secretion in the developing nervous system.[48] If anaesthetic-induced suppression of electrophysiological activity occurs during critical developmental periods, neurones that are pharmacologically 'disconnected' from the network might be pruned through apoptotic mechanisms.
Human Studies: There are several human cohort studies that suggest an association between early exposure to anesthetics and poor cognitive performance in later life. [49, 45, 50, 46, 51, 52, 47, 8] Those children receiving anesthesia before 3 years of age are more likely to have learning and behavior disorders compared with peers without anesthesia.[53, 54] Exposure to anesthesia in early life more than once or for a prolonged period adversely affects long-term neurodevelopmental outcomes in children.[45, 55]   Based on these studies it may be reasonable to speculate that neuropathological changes observed in the developing brain of animals similarly occur in brains of infants and children after anesthesia. However, coexisting conditions (low birth weight, medical problem, and especially surgical trauma) may preclude verification of the effect of anesthesia on cognitive development in human for ethical reasons. An ongoing study that will attempt to separate the effects of general anaesthesia from the surgical procedure is the GAS study (A Multi-site Randomized Controlled Trial Comparing Regional and General Anesthesia for Effects on Neurodevelopmental Outcome and Apnoea in Infants). of infants requiring inguinal herniorrhaphy.[56] A second study [Pediatric Anesthesia NeuroDevelopmental Assessment Study (PANDAS)] has undergone a feasibility study and is currently in the late planning stages to compare a retrospective cohort of children who received anaesthesia at less than 3 yr of age with unanaesthetized siblings in a prospective assessment of neurocognitive outcome in an attempt to reduce genetic and environmental contributions to cognitive performance.[57]
Methods of protection against anesthetic-induced neurotoxicity - their cellular and molecular mechanisms of neuroprotection
The possibility of anesthetic-induced neurotoxicity raises concerns regarding current anesthesia practice for pregnant women, infants and children. Accordingly, it is essential to develop and explore clinically relevant neuroprotective strategies in animals.
Erythropoetin (EPO): Epo could have a direct neurotrophic and neuroprotective effect, particularly in conditions of neural damage Influence the release of neurotransmitters, playing an important role in synaptic plasticity.[58]   Anesthetics may inhibit EPO production, resulting in neurotoxicity.[59] Role of EPO needs to be established.
Brain Preconditioning with Anesthetics:  Prior exposure to low dose of anesthesia, or a shorter duration of anesthetic exposure, can attenuate injury from high dose or prolonged anesthetic exposure in the developing brain.[60] Preconditioning with isoflurane[61] , propofol[62] and ketamine can protect from anesthetic-induced neurotoxicity. Pretreatment with inertia gas anesthetic xenon can also attenuate anesthetic induced neurotoxicity.[63] Xenon’s neuroprotective effect may be through its ability to inhibit intrinsic and common apoptotic pathways.[64] On the other hand, the anesthetic nitrous oxide or hypoxia pretreatment cannot protect from anesthetic-induced neuroapoptosis and cognitive function impairment.
Vitamins:
Nicotinamide : Single dose of 1 mg/kg nicotinamide attenuates ketamine-induced neuronal cell loss in the developing rat brain.[65] This reduced neuroapoptosis involves downregulation of Bax, inhibition of cytochrome c release from mitochondria into cytosol, and reduction in activated caspase-3 levels. Nicotinamide is also a potent inhibitor of proinflammatory cytokines. It may inhibit isoflurane-induced increase in levels of proinflammatarory factors TNFα ,IL-6, and IL-1β, thus protecting from neurodevelopmental disorders. Vitamin D3 (1-α-2,5-dihydroxy-vitamin D3) can also protect against ketamine-induced neuroapoptosis. Vitamin D3 can induce CaBP expression or enhance trophic factor action, both of which can stabilize intracellular calcium.[66] Vitamin C, known as an antioxidant[67] may also be effective against anesthetic-induced neurotoxicity.
Alpha2(α2) Adrenoceptor Agonist: α2 adrenoceptor signaling plays a trophic role during neurodevelopment and is neuroprotective in several settings of neuronal injury. Dexmedetomidine neuroprotection appears to involve a decrease in cleaved caspase-3 levels, and reversal of isoflurane-induced decrease in anti-apoptotic Bcl-1, pERK1, and pERK2 protein expression in vivo.[68] Neuroprotective mechanisms of α2 adrenoceptor signaling also involve inhibition of calcium entry, scavenging of glutamate, and reduction in NMDA receptor activation.[69]
Lithium: lithium, as a  GSK-3β inhibitor (glycogen synthase kinase-3β), has shown protective effects against neuroapoptosis induced by drugs.[70] Lithium treatment can also significantly increase BDNF serum levels, and suppress neuroapoptosis in central nervous system through the BDNF-Akt-Bcl2 antiapoptotic signaling pathway
Activity-Dependent Neuroprotective Protein (ADNP): Injection of NAP can inhit anesthetic induced caspase levels in dose dependent manner.[71]
Melatonin: Neuroprotective effect may be mediated by inhibition of mitochondria-dependent apoptotic pathway[72] .
Acetyl-L-carnitine:  metabolized in the brain to acetyl coenzyme A which subsequently enters the tricarboxylic acid cycle. It has been found to effectively block neuronal apoptosis caused by exposure to a combination of N2O and isoflurane.[73]
Conclusion: The evidence for anesthesia-induced neurodegeneration in animal models is compelling  and has suggested the possibility for deleterious effects of anesthetics in pediatric patients. All currently clinically utilized anesthetic drugs have been found to induce neuronal cell death in the developing brain and to potentially cause long-term neurological impairment. The exact molecular mechanisms neuronal toxicity of general anesthetics and sedatives on developing brain is not completely understood. Conversely, painful stimuli without analgesia and anesthesia have also been shown to initiate a harmful stress response in young children and to trigger neurotoxic effects in the developing brain, which can be blunted by anesthetics.  The mechanisms and human applicability of anesthetic neurotoxicity and neuroprotection remain under intense investigation.