Cell cycle activation contributes to isoflurane‐induced neurotoxicity in the developing brain and the protective effect of CR8

Abstract Aims It is well established that exposure of common anesthetic isoflurane in early life can induce neuronal apoptosis and long‐lasting cognitive deficit, but the underlying mechanisms were not well understood. The cell cycle protein Cyclin B1 plays an important role in the survival of postmitotic neurons. In the present study, we investigated whether cyclin B1‐mediated cell cycle activation pathway is a contributing factor in developmental isoflurane neurotoxicity. Methods Postnatal day 7 mice were exposed to 1.2% isoflurane for 6 hours. CR8 (a selective inhibitor of cyclin‐dependent kinases) was applied before isoflurane treatment. Brain samples were collected 6 hours after discontinuation of isoflurane, for determination of neurodegenerative biomarkers and cell cycle biomarkers. Results We found that isoflurane exposure leads to upregulated expression of cell cycle‐related biomarkers Cyclin B1, Phospho‐CDK1(Thr‐161), Phospho‐n‐myc and downregulated Phospho‐CDK1 (Tyr‐15). In addition, isoflurane induced increase in Bcl‐xL phosphorylation, cytochrome c release, and caspase‐3 activation that resulted in neuronal cell death. Systemic administration of CR8 attenuated isoflurane‐induced cell cycle activation and neurodegeneration. Conclusion These findings suggest the role of cell cycle activation to be a pathophysiological mechanism for isoflurane‐induced apoptotic cell death and that treatment with cell cycle inhibitors may provide a possible therapeutic target for prevention of developmental anesthetic neurotoxicity.


| INTRODUC TI ON
Millions of children undergo surgical operations or diagnostic procedures with general anesthesia across the world each year. 1 Over the past decade, an increasing number of reports from preclinical investigations have found that exposure to common anesthetic agents (eg, isoflurane and sevoflurane) during vulnerable periods of brain development results in widespread cellular toxicity including apoptosis and neurodegeneration, which in turn leads to neurocognitive impairment and decreased neuronal density in adults. 1,2 Among the clinically used general anesthetic drugs, isoflurane is more likely to cause widespread neurodegeneration in the newborn rodents, pigs, or non-human primates. [3][4][5][6] But the specific molecular mechanisms and pathways of isoflurane-induced neurotoxicity in the developing brain still remain to be elucidated.
In proliferating cells, the cell cycle is governed by a complex network of signaling pathways, and progression through different phases requires sequential activation of a large group of cell cycle regulatory molecules. 7 Neurons, usually regarded as postmitotic cells, are considered to arrest in the G0 phase and unable to proliferate. 8 Recent studies have suggested a relationship between aberrant cell cycle progression and neuronal apoptosis in the developing nervous system, as well as neural degeneration in distinct models of adult brain following stroke and trauma, epilepsy, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. [9][10][11][12][13][14][15][16][17] Cell cycle inhibition offers neuroprotection both in vivo and in vitro. [18][19][20][21][22] Cyclin B1 is a cell cycle protein involved in the G2/M transition and plays a vital role in the survival of postmitotic neurons. 23 In healthy neurons, cyclin B1 degradation takes place in the proteasome after ubiquitylation via the complex/cyclosome (APC/C)-cadherin 1 (Cdh1). 24 In the pathophysiological situation of Alzheimer's disease, 25 experimental brain ischemia, 26 traumatic brain injury, 19 however, the affected brain regions aberrantly express cyclin B1. Accumulation of cyclin B1 leads to activation of cyclin B1-Cdk1(cyclin-dependent kinase 1) complex, which is responsible for the increased phosphorylation of Bcl-xl and Bcl-2, and contributes to subsequent activation of the intrinsic cell death pathway 23,27 Although previous studies have identified cyclin B1/ Bcl-X L as a key pathway in neuronal apoptosis, the potential role of this signal transduction pathway in the regulation of neuronal cell death following isoflurane anesthesia in developing brain has not been addressed.
We hypothesized that isoflurane induces cyclin B1-mediated cell cycle activation, contributing to general anesthesia related neuronal cell death in the developing brain, and that inhibiting this pathway by a novel potent selective CDK inhibitor, CR8, reduces isoflurane-induced neuronal cell death. In the present study, we tested these two hypotheses, using a well-established experimental model of isoflurane-induced neurotoxicity in neonatal mice.

| Animals and experimental procedure
All the animal protocols were approved by the Institutional Animal Care and Use Committees. C57BL/6 mice (n = 45, obtained from Model Animal Research Center of Nanjing University, Nanjing) were housed with food and water available ad libitum and maintained under defined protocol with a 12 hours light-dark cycle.
In the isoflurane exposure study, mice at postnatal day 7 (P7) were placed inside a clear plexiglass chamber, resting on a heating pad to maintain their body temperature at about 37°C, and received 1.2% isoflurane in 30% oxygen/air at 5 L/min for 6 hours continuously. Mice in the control group were exposed to 30% oxygen/air only for 6 hours at room temperature. In the CR8 treatment study, mice were intraperitoneally administered 1 mg/kg of CR8 or an equal amount of vehicle 30 minutes before exposure to isoflurane.
The dose of CR8 was selected based on previous in vivo study showing brain neuroprotection of CR8 after spinal cord injury. 28 The concentration of isoflurane, oxygen, and carbon dioxide in the chamber was continuously monitored using an anesthetic agent analyzer. All animals were closely inspected for respiratory effort and skin color.
Isoflurane treatment of up to 1.5% for 6 hours had been shown not to cause hypoxia in our pilot study as well as previous report. 5

| Tissue collection and western blot
At 6 hours after discontinuation of isoflurane, mouse pups were euthanized; the brains were immediately removed; and the cerebral cortices

| Tissue processing and immunohistochemistry
At 6 hours after discontinuation of isoflurane, the mice were euthanized with deep anesthesia and perfused transcardially with saline followed by 4% paraformaldehyde in Tris-buffered saline. in the cerebral cortex, as well as hippocampus of each brain slice were counted by a blinded examiner. A total of four sections were counted per animal.

| Statistical analyses
All data are presented as the mean ± SEM. Statistical analyses were performed with the GraphPad Prism 5 software using Student's ttest for unpaired data or one-way ANOVA with the Newman-Keuls post hoc test whenever appropriate. P < 0.05 were regarded statistically significant.

| Postnatal exposure to isoflurane induced brain caspase-3 activation
Caspase-3 activation plays a central role in the process of apoptosis, and the cleavage of caspase-3 is a well-established biomarker for cell death by apoptosis. We therefore set out to determine the expression of cleaved caspase-3 positive cells in the brain tissues of postnatal day 7(P7) mouse pups receiving either air/oxygen or 1.2% isoflurane for 6 hours. As shown in Figure 1A-C, the number of cleaved caspase-3 cell was 13 ± 2/mm 2 when mouse pups were exposed to air/oxygen; however, administration of 1.2% isoflurane for 6 hours resulted in a significant increase of cleaved caspase-3 expression (122 ± 7/mm 2 ; P < 0.0001 vs sham). Similarly, immunoblotting revealed that isoflurane exposure led to a 239% (P < 0.0001) increase in cleaved caspase-3 protein levels at 6 hours after anesthesia ( Figure 1D, E). Thus, the changes of cleaved caspase-3 expression with both immunohistochemistry and immunoblotting assay validated that isoflurane induced significant apoptosis in the neonatal mouse brain.

| Isoflurane induced cell cycle-related protein activation
Continuous degradation of cyclin B1 and inactivation of CDK1/cyclin B1 complex are essential for the survival of postmitotic neurons.

| CR8 Inhibits cell cycle and caspase-3 activation in the cortical tissue after isoflurane exposure
In considering that isoflurane may induce apoptosis through regulating the cell cycle-related protein activation, we next investigated whether the isoflurane-induced neurotoxicity can be reversed by pharmacological cell cycle inhibitor. CR8 is a selective CDK inhibitor that has been shown to attenuate neuronal cell cycle activation and neurodegeneration in a mouse isolated thoracic spinal cord contusion model. 28 We therefore set out to determine the above activated cell cycle markers in isoflurane-treated mouse pups with or without CR8 pretreatment.
Notably Cyclin B1/Cdk1 is capable of phosphorylating the B-cell lymphoma extra-large (Bcl-xL) and induces antiapoptotic response. 23 We further assessed the effect of CR8 on apoptotic-related protein expression.
As shown in Figure 3 , and phospho-n-myc (F) in the mouse brain after exposure to isoflurane or 30% oxygen/air, normalized to GAPDH levels. Data presented are mean ± SEM compared with sham (n = 4-6, *P < 0.05, ***P < 0.001) compared to the control condition, which can be attenuated by CR8 administration ( Figure 3A, G P < 0.001 vs vehicle).

| Isoflurane-induced Cyclin B1 immunostaining positive cells are mainly neurons
Since cell cycle proteins are present in each cell type, and neurons are terminally differentiated, we then investigated if neurons were mainly involved in isoflurane-induced cell cycle activation using immunofluorescence analysis. As can be seen in Figure 4,column I is the immunofluorescence imaging of cyclin B1 (green), column II is the im-

| CR8 inhibits isoflurane-induced degeneration of neurons in the brain
To examine the potential protective effect of CR8 on isoflurane neurotoxicity at histological level, Fluoro-Jade B stainings of the mice brain at 6 hours after discontinuation of isoflurane were performed. Representative immunofluorescence images were shown in

| D ISCUSS I ON
In the current study, we observed that exposure to a commonly used general anesthetic isoflurane for 6 hours caused substantial neuronal apoptosis in the postnatal day 7 mouse brain, which involves blasts. 31 Notably, cyclin B1 has been found to accumulate in degenerating neurons of experimental cerebral ischemia and traumatic brain injury, as well as in patients' brain with stroke and Alzheimer's disease. 19,25,26 In postmitotic neurons, excitotoxic stimulation triggers neuronal apoptotic death through cyclin B1 accumulation in the nucleus. 23 We therefore reasoned that inappropriate cyclin B1 upregulation may be involved in isoflurane-induced neuronal degeneration.
The cyclin B1-Cdk1 complex in the mitochondria acts upstream of antiapoptotic protein Bcl-xL. 23 The Bcl-xL is a member of the Bcl-2 family, which plays a critical role in promoting cell survival by regulating mitochondrial membrane permeabilization. 29 Conditional deletion of Bcl-xL in the brain induces apoptosis in the upper layer cortical neurons at the early postnatal stages. 32  pairments. 35 In another study, 7-day-old mice with 0.75% isoflurane for 6 hours induces significantly increased apoptosis cell death without significant change in cell cycle regulatory proteins (CDK4, cyclin D1). 5 These findings suggest that isoflurane may require threshold concentration to induce neuronal cell cycle activation and that aberrant cell cycle reentry is another pathway rather than the primary mediator of isoflurane-induced neuronal apoptosis. 34 The current study evaluated the neuroprotective efficacy of a pharmacological cyclin-dependent kinases (CDKs) inhibitor CR8.
CR8 is a N6-biaryl-substituted derivative of roscovitine, which has been shown to remarkably alleviate neurodegeneration, learning and memory impairment induced by postnatal isoflurane exposure. 35 However, the therapeutic potential of roscovitine is confined by rapid metabolic deactivation and a short biological half-life. 36 In addition, its potency for inhibition of purified CDKs and CDK activity in cell lines is relatively weak. CR8 has enhanced inhibition of purified CDK1/2/3/5/7/9, improved solubility, cell permeability, and intracellular stability, leading to about 68-fold more potency than roscovitine in various cell lines in vitro. 36 Studies have demonstrated that CR8 significantly attenuate neuronal cell cycle activation and progressive neurodegeneration in multiple models of experimental traumatic brain injury (TBI) and spinal cord injury. 19,[36][37][38] In conclusion, our results identify a cascade of events triggered by isoflurane exposure in the developing brain. This transduction pathway includes upregulation of neuronal cell cycle protein cyclin B1, activation of Cdk1, and phosphorylation of antiapoptotic protein Bcl-xL. Phosphorylated Bcl-xL initiates cytochrome c release and caspase-3 activation that results in apoptotic cell death.
Furthermore, we demonstrated for the first time that the selective CDKs inhibitor CR8 confers protection against isoflurane-induced cell cycle activation and neurodegeneration. These findings suggest that use of cell cycle inhibitors may provide a possible therapeutic target for prevention of developmental anesthetic neurotoxicity.

ACK N OWLED G M ENTS
This study was supported by Natural Science Foundation of China (grant no. 81271205) and Foundation of Shenzhen science and technology innovation(grant no. 201703073000395).
F I G U R E 5 CR8 inhibits isofluraneinduced degeneration of neurons in the postnatal mouse brain. (A-C) Immunofluorescence photomicrographs of Fluoro-Jade B (green) positive cells in mouse brain exposed to 1.2% isoflurane with or without CR8 (scale bar = 100 μm).