N-Butylphthalide Improves Cognitive Function in Rats after Carbon Monoxide Poisoning

Abstract

Cognitive impairment is the most common neurologic sequelae after carbon monoxide (CO) poisoning, and the previous investigations have demonstrated that N-Butylphthalide (NBP) could exert a broad spectrum of neuroprotective properties. The current study aimed to investigate the effect of NBP on cognitive dysfunction in rats after acute severe CO poisoning. Rats were randomly divided into a normal control group, a CO poisoning group and a CO+NBP group. The animal model of CO poisoning was established by exposure to CO in a chamber, and then all rats received hyperbaric oxygen therapy once daily, while rats in CO+NBP group were administered orally NBP (6 mg/ 100g) by gavage twice a day additionally. The results indicated that CO poisoning could induce cognitive impairment. The ultrastructure of hippocampus was seriously damaged under transmission electron microscopy, and the expressions of calpain 1 and CaMK II proteins were significantly elevated after CO exposure according to the analysis of immunofluorescence staining and western blot. NBP treatment could evidently improve cognitive function, and maintain ultrastructure integrity of hippocampus. The expression levels of both calpain 1 and CaMK II proteins in CO+NBP group were considerably lower than that of CO poisoning group (P < 0.05). Taken together, this study highlights the molecular mechanism of cognitive dysfunction in rats after CO exposure via the upregulation of both calpain 1 and CaMK II proteins. The administration of NBP could balance the expressions of calpain 1 and CaMK II proteins and improve cognitive function through maintaining ultrastructural integrity of hippocampus, and thus may play a neuroprotective role in brain tissue in rats with CO poisoning.

Keywords: CO poisoning; Ca2+/calmodulin dependent protein kinase II; Calpain 1; N-butylphthalide; cognitive function; rat.

FIGURE 1

The average ratio of active avoidance response (AAR) in NC, CO poisoning and CO+NBP treatment groups. The average ratio of AAR in rats in CO group was notably decreased compared with those in NC group (n = 4, ^∗P < 0.05). After administration of NBP, the average ratio of AAR was significantly increased, especially at a late stage of CO poisoning (>7 days), and accompanied by a statistical significance from 7 days to 1 month after CO poisoning compared to that of CO group (n = 4, ^#P < 0.05). F = 11.528∼20.176.

FIGURE 2

Pathological changes of hippocampus in NC, CO poisoning and CO+NBP treatment groups using HE staining. Neurons in CA1 region were smaller accompanied by either round or oval shape, and were aligned neatly and tightly (A1), while those in CA3 (A2) were larger than in CA1 region and did not arrange in order. Meanwhile, few nuclear karyopyknosis and fragmentation were detected in NC samples. Neurons in both CA1 (B1) and CA3 (B2) regions in CO group were irregular shape, and part of which was pyknosis and shrinking with spindle shape. Neuronal body was roughly normal in both CA1 (C1) and CA3 (C2) regions in CO+NBP group.

FIGURE 3

Transmission electron microscopy (TEM) photographs of pathological changes in hippocampus in different groups. The exterior contour of neurons in NC group was clear, with big and round nucleus and uniform chromatin on days 3 (A1). The double nucleus membrane was clear and complete. Organelles were rich and scattered in cytoplasm with structural integrity (A2). Hippocampal neurons were swelled, chromatin in the nucleus was condensed and marginalized, and cristae and membrane were broken. Meanwhile, mitochondria appeared to be vacuolization, and partial cell organelle dissoluted and disappeared in CO poisoning group on days 3 (B1,B2). By contrast, the double-deckered nuclear membrane of neuron was clear, and mitochondria and other organelles were normal or only slightly damaged in CO+NBP group on days 3 (C1,C2).

FIGURE 4

Histogram of the number of dendrite spines per 10 μm in each group using Golgi staining. The number of dendritic spines in hippocampal neurons in CO group was lower than that of NC group, but no statistical difference was detected at an early stage of CO poisoning compared with that of NC group (<1 week, P > 0.05), whereas a significant difference existed at a late stage of CO poisoning (>2 weeks, ^∗P < 0.05). The administration of NBP could increase the number of dendritic spines, and it existed statistical difference as compared with CO group at a late stage of CO exposure (>2 weeks, ^#P < 0.05). F = 13.327∼19.460.

FIGURE 5

Changes of calpain 1 in hippocampus in each group. Calpain 1 positive cells were observed in hippocampal tissue in NC group, and mainly located in cytoplasm (A1–C4). After exposure to CO, the amount of calpain 1 positive cells was gradually increased, peaked between days 3 and 7, and maintained at relatively higher levels till 2 weeks in contrast to those in NC group (n = 4, P < 0.05). In contrast, administration of NBP could significantly decrease the expression levels of calpain 1 protein in comparison to that of CO poisoning group at the same time (n = 4, P < 0.05). (D) Histogram of the OD values of calpain 1 positive cells in each group at different time points.^∗Compared with NC group (n = 4, P < 0.05); ^#compared with CO group (n = 4, P < 0.05). F = 11.525∼21.618. Scale bar is 30 μm.

FIGURE 6

Expressions of CaMK II, calpain 1 and β-actin proteins using western blot assay. (A) The expression changes of CaMK II, calpain 1 and β-actin proteins in each group; (B) the relative A value of calpain 1 protein in different group at given time points; (C): the relative A value of CaMK II protein in each group in different time points. ^∗ VS. NS group, P < 0.05; ^# VS. CO group, P < 0.05, F = 12.436∼30.150.

FIGURE 7

Alterations of CamK II positive cells in each group. CaMK II positive cells were observed in rats in NC group (A1–A4), while the amount of CaMK II positive cells in CO poisoning individuals was sharply increased to the peak between days 1 and 3 with strong fluorescent values, and then decreased (B1–B4). Nevertheless, it was still higher than that of NC group till 1 month (n = 4, P < 0.05). NBP administration could down-regulate the expression level of CamK II compared with that in CO group at the same times (C1–C4; n = 4, P < 0.05). (D) Histogram of the OD values of CaMK II positive cells in each group at different time points. ^∗Compared with NC group (n = 4, P < 0.05); ^#compared with CO group (n = 4, P < 0.05). F = 10.941∼22.437. Scale bar is 30 μm.

FIGURE 8

Photographs of relationship between the locations of calpain 1 and CaMK II proteins in hippocampus under a fluorescent microscope. (A) Calpain 1 positive cells; (B) CaMK II positive cells; (C) co-expressions of the two proteins under the same view (merged). Scale bar is 30 μm.

FIGURE 9

A schematic diagram of the role of calpain 1 and CaMK II in apoptosis and long-term potentiation (LTP) after CO poisoning. Under resting state conditions, NMDARs were interacted with magnesium ion and were not activated, the level of intracellular calcium ions was relatively low, and there was only a small amount of calpain 1 and CaMK II proteins in cytoplasm to maintain the structural and functional integrity of cells. Under the pathological circumstances, such as a strong or persistent electrical stimulation, ischemia and hypoxia, acute severe CO poisoning, NMDARs were completely activated, and the overexpressions of calpain 1 and CaMK II proteins in cytoplasm induced by calcium overload were rush into nucleus, thus participated in the process of LTP and apoptosis or necrosis.