Effects Of Zinc Deficiency On Learning And Memory In Mice Of Alzheimer’s Disease Undergoing Isoflurane Anesthesia And Its Mechanisms

Background: Alzheimer’s disease (AD), also known as senile dementia, is a commonprogressive neurodegenerative disorder which accounts for the most frequent cause of humandementia and yet no effective therapy has been found to date. The prevalence of AD increasedfrom affecting12%of people over65years old to50%of people over85; the prevalencedoubles every five years after the age of60. AD has currently become one of the mostchallenging problem to the geratology. Along with up-to-date theory of anesthesia andimproving anesthesia techniques, an increasing number of surgeries were performed undergeneral anesthesia for the senile patients. However, it comes up with a new concerningquestion about the effects of general anesthesia on the senile patients and/or the patients withneurodegenerative disorders such as AD and Parkinson’s disease (PD). This is amuch-talked-about topic in the realm of anesthesiology research for the past few years. Thecause of AD has not been elucidated and it is reported that genetic and environmental factorsmay contribute to the etiology of AD, with aging the most important. Up-to-dateepidemiologic research and large quantities of studies evidenced that general anesthetics wasalso a precipitating factor of AD pathogenesis, it increased the risk of AD and aggravated ADneuronal pathology. General anesthesia has been reported to decrease the age of onset andincrease the incidence of AD. Compared with local anesthesia, general anesthesiasignificantly increased the risk of AD pathology in patients with cardiac surgery. Studies haveshown that general anesthesia aggravates the symptoms of AD and the neuronal pathology. Itis estimated recently that more than23million people worldwide suffered from anesthesiaand surgery every year, with AD patients undergoing anesthesia for more than8million. Theonset of AD is of asymptomatic and it may experience a pre-symptomatic period for15to30years (pre-clinic period) during which might be a certain amount of patients undergoinggeneral anesthesia and these amounts of patients will be presented with a huge risk ofanesthesia security. Therefore, it is imperative for us to understand the mechanisms of general anesthetics on AD pathogenesis and the risk factors as well as therapeutic strategies for AD.Zinc is the second but most important trace element of human body, it is essential fornormal brain function particularly in learning and memory. Zinc plays critical roles incombination with a variety of metalloproteinase activity sites and regulates thesemetalloproteinase as well as brain function in the form of neurotransmitters which are veryimportant in learning and memory. It is estimated that more than25%of the AD patientsworldwide suffered from zinc deficiency and presented with a lowered concentration of serumzinc which is closely related to cognitive dysfunction. Studies have shown that zincdeficiency aggravated neuronal pathology and cognitive dysfunction in an animal of AD. Wefound that zinc dyshomeostasis (zinc deficiency) contributed to severe damage of neurons,leading to the onset and progression of neurodegenerative diseases such as AD. It is reportedthat the most commonly used general anesthetics isoflurane is of neurotoxicity with AD andlong time or repeated exposure is associated with acceleration and progression of ADpathology. Animal studies have demonstrated that clinical relevant concentration of isofluraneinduces neuronal apoptosis, accelerates beta-amyloid protein (A) aggregation, aggravatesAD pathology and results in congnitive dysfunction and brain damage as is shown in AD.However, no evidence shows that zinc deficiency is a major risk factor for the onset of ADand it is not elucidated whether zinc deficiency aggravates isoflurane-mediated neurotoxicityand learning and memory impairment.Purpose:(1) To explore the effects of zinc deficiency on learning and memory functionin mice of Alzheimer’s disease undergoing isoflurane anesthesia and to discuss itsmechanisms.(2) To explore the role of zinc deficiency in isoflurane-mediated ADpathogenesis and to provide experimental evidence for clinical anesthetics and anesthesiaprotocol in patients of zinc-deficient AD and cognitive dysfunction.Methods: Eight-month old APP/PS1transgenic mice were randomly assigned to fourgroups (n=20): Zinc-replete group (Control group), Zinc-replete mice with Isoflurane (Isogroup), Zinc-deficient group (Zinc group), Zinc-deficient mice with Isoflurane (Iso+Zincgroup). In the control group, mice were given normal zinc-replete diet (Zn:30mg/kg diet)for1month; In the Iso group, mice were given normal zinc-replete diet (Zn:30mg/kg diet) for1month and then they were exposed to isoflurane1.4%for2h; In the Zinc group, micewere given zinc-deficient diet (Zn:1mg/kg diet) for1month; In the Iso+Zinc group, micewere given zinc-deficient diet (Zn:1mg/kg diet) for1month and then they were exposed toisoflurane1.4%for2h. Before exposure to isoflurane, serum zinc concentration of all micewas measured by Inductively coupled plasma mass spectrometry (ICP-MS) to make sure thezinc-deficient animal model was successfully built (those mice of unsuccessful zinc-deficientanimal model were excluded). Half of the mice were used for behavioral tests and the otherhalf were used for Terminal deoxynucleotidyl transferase-mediated deoxy-UTPfluoresceinnick end labeling (TUNEL) staining and western blot analysis. Spatial learning and memoryfunction was tested with Morris Water Maze (MWM)24h after isoflurane exposure. TUNELstaining was performed for hippocampal neuronal apoptosis. Western blot analysis was usedto detect aggregation of A (A, A1-40, A1-42), phosphorylation of hippocampal tauprotein at the site of Ser262(pS262), Thr231(pT231) and Ser396(pS396) as well asphosphorylation of total tau protein (p-tau) levels, variation of mitochondrial mediatedneurons apoptosis genes such as Bcl-2, Bax, Caspase-3and endoplasmic reticulum (ER)stress mediated neurons apoptosis genes such as CHOP, Caspase-12, Grp78.Results:(1) Zinc-deficient animal model was successfully built. Serum zinc levels in thecontrol group and Iso group were normal. Compared with control group, serum zinc levels inthe Zinc group and Iso+Zinc group decreased significantly, which indicated successfulbuilding of zinc-deficient animal model.(2) Effects of zinc deficiency on learning andmemory in APP/PS1transgenic mice undergoing isoflurane anesthesia. No differences ofescape latency and spatial exploring time were found between the control group and Iso group,indicating that1.4%isoflurane for2h did not impair learning and memory in APP/PS1transgenic mice. In the Zinc group, the mice showed longer latency to locate the hiddenplatform and shorter spatial exploring time than the mice in control group, suggesting thatzinc deficiency impaired learning and memory functions in APP/PS1transgenic mice.Compared with Zinc group, the mice showed significant longer escape latency and shorterspatial exploring time, suggesting that zinc deficiency aggravated learning and memoryfunctions in APP/PS1transgenic mice exposed to isoflurane.(3) Effects of zinc deficiency on hippocampal neuronal apoptosis in APP/PS1transgenic mice undergoing isofluraneanesthesia. Compared with control group, apoptosis of CA1hippocampal neuronal cellsincreased in the Iso group and Zinc group, respectively, indicating that both1.4%isofluranefor2h and zinc deficiency induced neuronal apoptosis of hippocampus in CA1region. Whenzinc-deficient mice were exposed to1.4%isoflurane for2h, apoptosis of CA1hippocampalneuronal cells increased significantly compared to that in Zinc group, suggesting that zincdeficiency aggravated isoflurane-mediated neuronal apoptosis of hippocampus in CA1region.(4) Effects of zinc deficiency on hippocampal A aggregation in APP/PS1transgenic miceundergoing isoflurane anesthesia. Isoflurane1.4%for2h did not enhance aggregation of A (including total A, A1-40and A1-42). Compared with control group, zinc deficiency didnot affect the aggregation of total A and A1-40, but the levels of A1-42increased,indicating that zinc deficiency induced deposits of A1-42, thus accelerated A pathology ofhippocampus in AD. We also found that when zinc-deficient mice were exposed to1.4%isoflurane for2h, the levels of total A and A1-40increased slightly but A1-42increasedsignificantly (p<0.05) compared to that in Zinc group, suggesting that zinc deficiencyaggravated A pathogenesis and A aggregation in hippocampus of APP/PS1transgenic micewhen exposed to1.4%isoflurane for2h.(5) Effects of zinc deficiency on hippocampal tauprotein phosphorylation in APP/PS1transgenic mice undergoing isoflurane anesthesia. Wefound no effects of1.4%isofluane2h on phosphorylation of hippocampal tau protein at thesite of Ser262(tau[pS262]), Thr231(tau[pT231]) and Ser396(tau[pS396]) as well asphosphorylation of total tau protein (p-Tau). The levels of total p-tau, pS262and pT231ingroup Zinc were elevated significantly compared to that in group control, whereas pS396elevated slightly, indicating that zinc deficiency did not alter the levels of pS396but enhancedphosphorylation of total p-Tau and pS262and pT231. When the mice of zinc deficiency wereexposed to1.4%isoflurane for2h, the levels of p-tau, pS262, pT231and pS396were allincreased significantly compared to that in group Zinc. These results suggests that zincdeficiency significantly aggravated the levels of total p-tau, pS262and pT231in APP/PS1transgenic mice when exposed to1.4%isoflurane for2h.(6) Effects of zinc deficiency onmitochondrial associated apoptosis signal pathway of hippocampus in APP/PS1transgenic mice undergoing isoflurane anesthesia. Compared with group control,1.4%isoflurane for2hincreased the levels of Caspase-3and Bax but decreased the levels of Bcl-2in APP/PS1transgenic mice, indicating that1.4%isoflurane for2h activated mitochondrial associatedapoptosis signal pathway through activation of Bax and Caspase-3and inhibition of Bcl-2. Inthe Zinc group, we found significantly increased levels of Caspase-3and Bax but decreasedlevels of Bcl-2in APP/PS1transgenic mice as compared to group control, suggesting thatzinc deficiency also activated mitochondrial associated apoptosis signal pathway throughactivation of Bax and Caspase-3and inhibition of Bcl-2. When zinc-deficient mice wereexposed to isoflurane, it showed significantly more increased levels of caspase-3and Bax buteven decreased levels of Bcl-2as compared to the mice in group Zinc. These findingssuggested that zinc-deficiency significantly aggravated mitochondrial impairment throughactivation of caspase-3and Bax and inhibition of Bcl-2in APP/PS1transgenic mice whenexposed to1.4%isoflurane for2h.(7) Effects of zinc deficiency on endoplasmic reticulumstress associated apoptosis signal pathway of hippocampus in APP/PS1transgenic miceundergoing isoflurane anesthesia. No changes were found with isoflurane exposure to themice of group control, suggesting that1.4%isoflurane for2h did not activate ER stressassociated apoptotic signal pathway. The levels of CHOP, Grp78and caspase-12in groupZinc were elevated significantly compared to that in group control, indicating that zincdeficiency activated ER stress associated apoptosis signal pathway through activation ofCHOP, Caspase-12and Grp78. When the mice of zinc deficiency were exposed to1.4%isoflurane2h, the levels of CHOP, Grp78and caspase-12were all increased significantlycompared to the mice in group Zinc. These results indicated that zinc-deficiency aggravatedactivation of ER stress through CHOP, Caspase-12and Grp78in APP/PS1transgenic micewhen exposed to1.4%isoflurane for2h.Conclusion: Isoflurane1.4%for2h does not impair learning and memory of APP/PS1transgenic mice, but it has been shown of potential neurotoxicity as it increased hippocampalneuronal apoptosis. Zinc deficiency significantly aggravated learning and memoryimpairment and neuronal apoptosis of hippocampus in APP/PS1transgenic mice exposed to1.4%isoflurane for2h. It is probably associated with hippocampal A aggregation and phosphorylation of tau protein, activation of mitochondrial and endoplasmic reticulum stressrelated apoptotic signal pathway.