Moderate-intensity interval training (MIT) can protect rats against Methamphetamine –induced injuries

Methamphetamine (METH) can cause neurotoxicity and increase the risk of neurodegenerative disorders such as Alzheimer disease and Parkinson disease. This study aimed to investigate the effect of moderate-intensity interval training (MIT) on gene expression and antioxidant status of the hippocampus of METH-dependent rats. Twenty-eight male Wistar rats were randomly divided into four equal groups (n=7): saline, METH, MIT, and METH+MIT. METH was injected intraperitoneally at 5 mg/kg for 21 days. The MIT (intermittent running) was performed on the treadmill 5 days a week for 8 weeks. Morris Water Maze test was performed to measure learning and memory. Then, the hippocampal tissue was extracted to evaluate changes in gene expression and biochemical enzymes. The data were analyzed using one-way and two-way ANOVA methods at P<0.05. The results showed that METH injection signicantly reduced spatial memory and antioxidant enzymes and increased the expression of α-synuclein (α-syn), cyclin-dependent kinase 5 (CDK5), tau and phosphorylated tau (p-Tau) genes compared to the saline group. MIT signicantly increased spatial memory and antioxidant enzymes. However, it reduced α-syn, CDK5, tau and p-tau expression. Thus, METH caused neural damage, and MIT could protect the neural system against METH-induced insults in male rats.


Introduction
Methamphetamine (METH), an illicit psychotropic drug, is abused by some people worldwide. It has been shown to increase dopamine secretion and regulate dopamine transport activity (DAT), thus selectively impairing the dopaminergic pathway in animal models (Prakash et al. 2017). METH consumers show various disorders in memory retrieval, verbal memory, and executive performance tests. Studies demonstrated that METH can cause hippocampal atrophy, neurodegeneration, and impaired memory and learning (Thompson et al. 2004). An animal study showed that hippocampal neurogenesis inhibition due to METH resulted in the development of cognitive and memory impairments (Recinto et al. 2012).
Research has indicated that oxidative stress is involved in altering the function of N-methyl aspartate (NMDA) receptors, inducing cell death, and activating microglia (Panenka et al. 2013). Certain posttranslational changes, such as phosphorylation, toxicity uptake, and alpha-synuclein (α-syn) nitrosillation, may lead to the accumulation of α-syn which is secreted by neurons and activates microglial cells.
Pathological α-syn and microglia activation by amplifying each other can lead to neuronal injury.
In addition, α-syn has been shown to activate astrocytes through Tol-like receptors (TLRs) 4 in vitro.
Dysfunction of synapses surrounded by astrocytes eventually leads to neural damage. It is hypothesized that METH-induced α-syn accumulation may directly lead to mitochondrial damage, myelin sheath destruction, and failure to form synaptic vesicles, and may indirectly lead to the overexpression of cyclindependent kinase 5 (CDK5) and glycogen synthase kinase 3 beta (GSK-3β). The activity of these kinases leads to tau phosphorylation and block autophagy (Rannikko et  There are several treatments for the neurotoxic effects of METH, one of them is voluntary exercise (sustained physical activity). It is considered as a physical activity intervention to reduce the destructive effects of METH. METH has been shown to interfere with normal brain function, and physical activity can prevent or reduce the disorder.

Exercise protocol
The exercises with moderate intensity (60% of maximum speed) were performed on a rodent treadmill at an inclination of 0° for 8 weeks, 5 days a week. The duration of training in each session was 36 minutes, including 6 minutes of warm-up with an intensity of 20% of maximum speed, 6 minutes of cooling with an intensity of 20% of maximum speed, and the main program of two sections of medium frequency with an intensity of 60% and 30% of the maximum speed (4 cycles) (Yazdanparast Chaharmahali et al. 2018).

Tissue sampling
After the last training session, the rats were anesthetized with CO 2 gas. Hippocampal tissue samples from the rats were isolated and immediately transferred to a -70°C freezer for biochemical and molecular tests.

Learning and memory test (Morris Water Maze)
The maze comprised a black circular tub (80 cm in height) lled with water (22±1°C) located in a room with special cues on the walls. A platform (8 cm in diameter) was located in the water (1.5 cm below the water surface). In the learning phase, the animals were trained 4 times a day for 4 days to nd the platform in the middle of one of the quarters of the maze. Each time the rat was randomly placed in water at one of the four quadrants (north, south, east, or west). If they failed to nd the platform, they would be placed on it for 10 s to associate its location with the spatial cues of the room. One day after the last learning trial, the animals underwent a probe trial to test their spatial memory. On the fth day, the rats were placed in a non-platform pool to check their spatial memory. For this purpose, the percentage of time spent in the target quadrant was recorded and analyzed. In both stages, the movements of the rats in the water were recorded by a camera positioned above the center of the water maze, and the data were collected by a computer equipped with the water maze software (Veschsanit et al. 2021).

Malondialdehyde (MDA) measurement
MDA is a compound that can be evaluated as a lipid peroxidation index. The levels of MDA were tested by the thiobarbituric acid (TBA) method. The absorbance of products was measured at 535 nm (Buege and Aust 1978).
Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity The activities of GPx and SOD were measured by using the Randox kits (UK; Cat NO.RS504, UK; Cat NO.SD125, respectively). The SOD activity was detected at the wavelength of 560 nm, and the GPx level was detected at the wavelength of 340 nm (Paglia and Valentine 1967).

Estimation of catalase (CAT) activity
CAT activity in the hippocampus was determined as described by Sinha (1972) with little modi cations.
The CAT activity was expressed as units/mg protein (Sinha 1972).

Total antioxidant capacity (TAC)
Hippocampal total antioxidant capacity was evaluated according to the method of Benzie and Strain (1996). In this method, the ability to eliminate added hydrogen peroxide was assessed. The remaining H 2 O 2 is measured using enzymatic reaction converting 3,5-dichloro-2-hydroxyl benzenesulfonate to a colored product determined at 532 nm (Benzie and Strain 1996 seconds. The primer sequences were designed by Primer-BLAST (NCBI) online software, and the gene (Gapdh) was used as an internal control gene (Table 1). Data analysis was performed based on threshold cycle comparison (CT). The ampli cation curve of each PCR reaction was normalized with the ampli cation curve of the corresponding Gapdh reference gene. The CT difference obtained from the tested and control samples was calculated, and the ratio of the target gene to the reference gene was calculated using the CT-2 formula.  (Figure 1). One way ANOVA test showed F (3, 20) = 7 p = 0.002.

MIT improves learning in the METH treated rats
To measure learning in the rst 4 days of the MWM test, the total distance traveled to nd the hidden platform was assessed. Our data did not show any signi cant difference between the groups, but in the rst 4 days of the learning phase, signi cant differences were observed within the groups. There was a signi cant difference in the saline group on the rst day with days 2, 3, and 4 (P = 0.009, P = 0.010, and P < 0.001, respectively). Furthermore, the METH group had a signi cant difference on the rst day with days 3 and 4 (P = 0.003 and P < 0.001, respectively). The METH + MIT group demonstrated a signi cant difference on the rst day with days 3 and 4 (P = 0.014 and P = 0.002); in addition, the second day also showed a signi cant difference with days 3 and 4 (P = 0.024 and P = 0.005, respectively). Finally, the rst day of the MIT group indicated a signi cant difference with days 2, 3, and 4 (P = 0.004, P = 0.026, and P = 0.004, respectively) ( Figure 2A).

MIT improves memory in the METH treated rats
There was a signi cant difference in the percentage of distance traveled (2.40±0.66) q(28)=8.41, p<0.

Discussion
Our results showed that METH administration in rats led to spatial memory impairment, reduced antioxidant enzymes, and increased the expression of α-syn, CDK5, Tau, and p-Tau genes. MIT signi cantly increased spatial memory and antioxidant enzymes, but signi cantly decreased the expression of the aforementioned genes.
In accordance with our result, Izawa et al. (2006) showed that METH reduced memory and induced confusion and amnesia, and its long-term use destroyed dopaminergic and serotonergic nerve endings in the brain (Izawa et al. 2006). In addition, Sertani et al. Our results demonstrated that antioxidant enzymes were reduced by METH; in other words, the antioxidant defense of hippocampal tissue against oxidative stress was greatly reduced. Kakita et al. (2002) showed that the use of METH in animal models causes oxidative stress and neurotoxicity due to the production of reactive oxygen species (Kakita et al. 2002). Hamakawa et al. (2013) reported that 3 weeks of exercise reduces the level of free radicals, which are associated with a decrease in oxidative damage and the resulting movement disorders (Hamakawa et al. 2013). In particular, Ogunovsky et al. (2005) demonstrated that moderate-intensity exercise (1 hour of swimming per day for 8 weeks) increases both antioxidant capacity and resistance to oxidative stress in the body. Note that over-training does not induce oxidative damage in the brain and does not cause loss of memory (Ogonovszky et al. 2005). It appears that METH injection results in the complex formation of CDK5 with P25, and the activity of this kinase is greatly increased, eventually leading to excessive Tau phosphorylation. MIT training possibly reduces the formation of this complex. Tau toxicity in Alzheimer disease is due to its deposition in the soma or dendrites of neurons or its high phosphorylation. It has been shown that CDK5 is activated by αsyn, and eventually, this kinase leads to tau phosphorylation. Various factors such as the toxic effect of β-amyloid deposition on the brain tissue and disruption of message transduction pathways due to hyperphosphorylation of tau protein are involved in the development of this disease. Ko et al. (2017) stated that endurance training increases Sirtuin 1 (SIRT-1) expression, which leads to increased mitochondrial biogenesis and reduced oxidative stress by the activation of peroxisome proliferatoractivated receptor-gamma coactivator 1-alpha (PGC-1α), and may improve mitochondrial function and autophagy process (Koo and Cho 2017). This study is consistent with the present study. It is possible that the level of PGC-1α in MIT training has increased; one of the limitations of the present study is the lack of PGC-1α measurement.

Conclusion
It seems that METH causes neurotoxicity in the hippocampal tissue, as well as decreased spatial memory and antioxidant enzymes by increasing oxidative stress at the cellar level. Increased α-syn, CDK5, Tau and p-Tau expression possibly leads to neuronal injury, and MIT may be effective against METH-induced injuries in male rats.
Con icts of interest/Competing interests: The authors hereby state that there is no con ict of interest in the present study.
Availability of data and material: The data will be available based on reasonable request.