Maternal Exposure of Rats to Isoflurane during Late Pregnancy Impairs Spatial Learning and Memory in the Offspring by Up-Regulating the Expression of Histone Deacetylase 2 - Fig 7

<p>HDAC2 inhibition reversed the downregulated expression of CREB mRNA caused by maternal isoflurane exposure: (a) Isoflurane exposure downregulated CREB mRNA expression. The CREB mRNA levels in the offspring hippocampus in isoflurane exposed group were lower than control group (* p < 0.05). With the increase of isoflurane exposure time, the downregulated effect became more obvious. The CREB mRNA levels in I8N group were higher than I2N group (^# p < 0.05). (b) SAHA reversed the down-regulation of CREB mRNA expression. Compared with relative non-SAHA subgroup, the levels of CREB mRNA in SAHA treated sugroup increased (* p < 0.05). But the CREB mRNA levels in I8S subgroup were still lower than normal control group (^# p < 0.05). This indicates that SAHA cannot completely reverse the downregulated effect caused by isoflurane when the exposure time prolonged to 8 hours.</p

HDAC2 inhibition reversed the overexpression of HDAC2 mRNA caused by maternal isoflurane exposure.

<p>The expression levels of HDAC2 mRNA in offspring hippocampus were detected by real time PCR (RT—PCR). The levels of mRNA were normalized to that of β-actin in the same sample and then normalized to the average values of control offspring in the same RT-PCR. The mean value of the mRNA expression level of all of the offspring born to the same mother rat was calculated as the final expression level of mRNA. (a) maternal isoflurane exposure potentiated the expression of HDAC2 mRNA. The HDAC2 mRNA levels in the offpsring hippocampus in I2N, I4N and I8N group were higher than normal control group (CN group, * p < 0.05). (b) SAHA reversed the overexpression of HDAC2 mRNA. The HDAC2 mRNA levels in SAHA treated subgroup were higher than non-SAHA subgroups (* p < 0.05).</p

HDAC2 inhibition alleviated the impaired learning caused by maternal isoflurane exposure.

<p>SAHA potentiates the learning ability of normal rats, the escape latencey in SAHA treated normal offspring was shorter than normal control offspring at 2^nd, 4^th and 5^th trial (* p < 0.05, Fig 3a); The escape latency in I2S, I4S and I8S group were shorter than their relative control groups (I2N, I4N and I8N group respectivly), but had no statistical differences (p > 0.05, Fig 3b, c and d). The escape latency in I8S group was longer than normal control group (CN group) at 3^rd, 4^th and 5^th trial (p < 0.05, Fig 3e). S = SAHA treated subgroup; N = non—SAHA treated subgroup.</p

The effect of isoflurane exposure on femoral venous blood gas and electrolytes in maternal rats.

<p>The effect of isoflurane exposure on femoral venous blood gas and electrolytes in maternal rats.</p

Maternal isoflurane exposure impaired learning and memory in offspring: Offspring of rats exposed to isoflurane on gestation day 18 (E18) for 2h (I2N), 4h (I4N) and 8h (I8N) respectively.

<p>Thirty days postneaonatal (P30), the learning and memory was assessed using the Morris water maze: (a) Escape latency (time to find the hidden platform). At the third trial, the escape latency in the I2N, I4N or I8N group was significant longer than the control group (^* p < 0.05); The escape latency increased with the increase of isoflurane exposure time. The escape latency in I8N group was significant longer than control group at 3^rd, 4^th, 5^th and 6^th trial (^▽ p < 0.05). At the 6^th training trial, the escape latency in the I8N group was significantly longer than the I2N or I4N group (^# p < 0.05); (b) Target quadrant traveling time. The offspring in isoflurane exposure group spent less time traveling in the platform hidden quadrant (target quadrant). The target quadrant traveling time in I8N group was significant less than CN group (^* p < 0.05), I2N and I4N group (^# p < 0.05); (c) Platform crossing times. The offspring in isoflurane exposure group swam across the location where the platform hidden (platform-crossing times) less than control group, especially those in I8N group. The platform-crossing times in I8N group was significant less than CN group (^* p < 0.05), I2N and I4N group (^# p < 0.05). CN = control group.</p

HDAC2 inhibition reversed the downregulated expression of NR2B caused by maternal isoflurane exposure.

<p>(A1) Amplification plot of NR2B and β-actin mRNA; (A2) Agarose gel electrophoresis images of NR2B and β-actin mRNA; (A3) Maternal isoflurane exposure downregulated the expression of NR2B mRNA (mean ± SE): The levels of NR2B mRNA in isoflurane exposure group (I2N, I4N, I8N) were lower than CN group (* p < 0.05). The NR2B mRNA levels in I4N and I8N group were lower than I2N group (^# p < 0.05); (A4) SAHA reversed the downregulated expression of NR2B mRNA (mean ± SE): The levels of NR2B mRNA in I2S, I4S and I8S subgroup were higher than I2N, I4N and I8N subgroup respectively (* p < 0.05). The NR2B mRNA levels in I8S subgroup were lower than CN group (^# p < 0.05). (B1) NR2B protein western blot images; (B2) Maternal isoflurane exposure downregulated the expression of NR2B protein (mean ± SD): The protein levels of NR2B protein were lower than CN group (^# compared with I2N group, p<0.05b); (B3) HDAC2 inhibition reversed the downregulated expression of NR2B protein: The protein levels of NR2B in I2S, I4S and I8S subgroups were higher than I2N, I4N and I8N sugroups respectively. The levels of NR2B protein in I8S subgroup were lower than CN group (^# p < 0.001). This indicates that SAHA cannot completely reverse the downregulated effect caused by isoflurane when the exposure time prolonged to 8 hours.</p

Experimental design.

<p>Pregnant dams were exposed to 1.5% isoflurane in 100% oxygen or to 100% oxygen alone for the times indicated on E18 and the offspring were treated with 90 mg/kg SAHA (ip) or vehicle (DMSO) 2 hours before behavioral testing. The number in parentheses represents the number of animals: F = female, M = male; DMSO = dimethyl sulfoxide; SAHA = suberanilohydroxamic acid, also known as vorinostat; TEM = transmission electron microscopy.</p

HDAC2 inhibition alleviated the hippocampal ultrastructure impairment caused by maternal isoflurane exposure (transmission electron microscopy, ×6000).

<p>The hippocampal ultrastructure showed apparent abnormality with the increase of isoflurane exposure time. The ultrastructure showed no differences compared to the control group when isoflurane exposure time was 2h (I2N). When isoflurane exposure time lengthen to 4h, the neuron number decreased, the nuclei became irregular, cytoplasmic area decreased, mitochondrial number decreased, and we observed evidence of disordered mitochondrial cristae. The quantity of rough endoplasmic reticulum, ribosome and Golgi apparatus decreased, and the ribosomes exhibited degranulation (I4N). When the exposure time prolonged to 8h, all of these changes became more prominent, there were fewer neurons with dilated intercellular space. Dissolved mitochondrial cristae and swollen Golgi apparatus could be observed (I8N). HDAC inhibition alleviated the impairments, but did not increase the neuronal number (I4S and I8S group).</p