3.1. General conditions and pre-existing cognitive impairment in aged mice was improved by resistance exercise
Mice were trained using a 1-meter ladder (Fig. 1a), body weight, food and water intake, as well as muscle strength were monitored during resistance training (Fig. 1b). Food and water intake did not differ between the active and sedentary groups (Fig. 1c). Aged mice were on average heavier than middle-aged mice, showing by an increased body weight baseline (5.23 ± 2.1g, p = 0.0205, Fig. 1). The body weight of both middle-aged and aged sedentary mice was significantly increased after 5 weeks (3.71 ± 0.5 g, p<0.0001 for 9M mice; 4.31 ± 0.5 g, p<0.0001 for 18M mice, Fig. 1d=. Resistance exercise reduced the body weight only in middle-aged but not older mice (-2.47 ± 0.65 g, p = 0.0024 for middle-aged mice; p = 0.3002 for aged mice, Fig. 1d), nevertheless, resistance exercise prevented weight gain otherwise seen in sedentary aged mice (interaction: F (1, 44) = 1.008, p = 0.3209; age factor: F (1, 44) = 4.132, p = 0.0481; exercise factor: F (1, 44) = 90.59, p < 0.0001. Multiple comparisons: -5.0 ± 0.85 g, p < 0.0001 for aged mice, Fig. 1e).
Recent studies suggest muscle strength can be used as an index to monitor progression of cognitive decline in old adults 29. Aged mice have significantly lower baseline muscle strength compared with their younger counterparts (-7.27 ± 1.46 points, p < 0.0001, Fig. 1f). Five weeks of resistance training significantly improved the weight lifting performance in mice from both groups (3.54 ± 1.39 points, p = 0.0256 for 9M mice; 7.99 ±1.18 points, p<0.0001 18M mice, Fig. 1f=. A similar trend was seen in further analysis of muscle strength change (interaction: F (1, 43) = 0.5549, p = 0.4604; age factor: F (1, 43) = 7.468, p = 0.0091; exercise factor: F (1, 43) = 25.71, p < 0.0001. Multiple comparisons: 5.538 ± 1.794, p = 0.0179 for 9M mice; 7.446 ± 1.828, p = 0.0011 for 18M mice, Fig. 1g). The effect of resistance exercise on learning and memory was evaluated by the Y maze test. We found a significant interaction between age and exercise, suggesting that the effect of resistance exercise differed between middle-aged mice and aged mice (for errors, interaction: F (1, 44) = 6.497, p = 0.0144; age factor: F (1, 44) = 46.59, p < 0.0001; exercise factor: F (1, 44) = 10.05, p = 0.0028, Fig. 1h. For latency, interaction: F (1, 44) = 2.301, p = 0.1364, age factor: F (1, 44) = 38.46, p < 0.0001; exercise factor: F (1, 44) = 5.838, p = 0.0199, Fig. 1i). Multiple comparisons found that aged mice demonstrated deficits in aversive memory and hippocampal-dependent spatial learning (errors: 5.1 ± 0.79, p < 0.0001, Fig. 1h; latency: 4.03 ± 0.75 s, p < 0.0001, Fig. 1i) when compared with the middle-aged mice. Resistance exercise reduced both errors (- 3.1 ± 0.79, p = 0.0015, Fig. 1h) and latency (- 2.05 ± 0.75 s, p = 0.0439, Fig. 1i) in aged mice.
Therefore, the current training protocol prevented the aged mice from becoming overweight without affecting their food and water intake, improved their muscle strength and cognitive function. Given that obesity30 and pre-existing cognitive impairment31 are independent risk factors for PNDs, our data indicate that resistance exercise might reduce the incidence of postoperative cognitive decline.
3.2. Resistance training improved postoperative cognitive performance
A laparotomy was performed 24h after the last resistance training session and the open field test and NOR tests were conducted on postoperative days 12 and 13(see Fig. 2a). As can be seen from figures 2b & 2c, postoperative locomotor activity was not affected in any of the groups. Surgery induced anxiety in mice from the laparotomy only group as indicated by a decreased central duration compared with control (interaction: F (1, 62) = 1.347, p = 0.2502; laparotomy factor: F (1, 62) = 7.808, p = 0.0069; exercise factors: F (1, 62) = 6.119, p = 0.0161. Multiple comparisons: -54.93 ± 19.04s; p = 0.0268 compared to control, Fig. 2d). Pre-operative resistance exercise appears to exert some anxiolytic effects, but the increased central duration time in the RE + Lap group failed to reach statistical significance (p = 0.0585 compared to laparotomy group, Fig. 2d).
Mice from the laparotomy group demonstrated the presence of recognition memory deficits following surgery as indicated by the significant decrease in discrimination index in the NOR test (interaction: F (1, 62) = 3.449, p = 0.0680; laparotomy factor: F (1, 62) = 11.6, p = 0.0012; exercise factor: F (1, 62) = 6.158, p = 0.0158. Multiple comparisons: -0.16 ± 0.04, p = 0.0016 compared to control, Fig. 2e). Similarly, they displayed more errors (interaction: F (1, 62) = 14.01, p = 0.0004; laparotomy factor: F (1, 62) = 5.778, p = 0.0192; exercise factor: F (1, 62) = 8.049, p = 0.0061. Multiple comparisons: 2.56 ± 0.57, p = 0.0002. Fig. 2f) and longer escape latency (interaction: F (1, 62) = 1.65, p = 0.2037; laparotomy factor: F (1, 62) = 6.092, p = 0.0164; exercise factor: F (1, 62) = 6.846, p = 0.0111. Multiple comparisons: 1.83 ± 0.67 s, p = 0.0392. Fig. 2g) compared to control in the Y-maze test. Preoperative resistance training attenuated these changes when compared to laparotomy only mice (discrimination index: 0.13 ± 0.04, p = 0.0162; errors: -2.7 ± 0.6, p < 0.0001; latency: -1.9 ± 0.7s, p = 0.0368, Fig. 2e-g).
3.3. Resistance training attenuated the decreased in dendritic process complexity and spine density following laparotomy
To evaluate whether the beneficial effect of resistance exercise on postoperative cognitive function was accompanied by structural improvements in neurons, we used Golgi-staining to show the morphology of dendrites and spines (Fig. 3a & 3b). The spine density was significantly decreased in the surgery group (interaction: F (1, 68) = 1.89, p = 0.1738; laparotomy factor: F (1, 68) = 10.96, p = 0.0015; exercise factor: F (1, 68) = 7.189, p = 0.0092. Multiple comparisons: -10.63 ± 3.21%, p = 0.0079, compared to control, Fig. 3c). Preoperative resistance training ameliorated this reduction (9.21 ± 3.21%, p = 0.0275, Fig. 3c). Sholl profiles generated by semi-automated analysis showed a reduction in dendritic crossings following surgery, indicative of decreased dendritic length and branching within 145 - 200µm of the radius (interaction: F (150, 2700) = 1.833, p < 0.0001; laparotomy factor: F (50, 2700) = 194.5, p < 0.0001; exercise factor: F (3, 54) = 7.736, p = 0.0002. Multiple comparisons: p < 0.05, p < 0.01, compared to control, Fig. 3d-f). Resistance exercise group had greater numbers of dendritic crossings within 115 - 210µm of the radius compared to laparotomy group (p < 0.05, p < 0.01, p < 0.001, p < 0.0001, compared to laparotomy group, Fig. 3d-f). Combining with the behavioral data, our result indicates that resistance exercise might be an effective strategy to prevent PNDs, as well as surgery-induced dendritic and synaptic deficit.
3.4. Preoperative pro-inflammatory disposition in aged mice and early inflammatory responses were reduced by resistance exercise
Both animal and human data suggest that ageing is commonly associated with chronic neuroinflammation “inflammaging”, which can be acute inflammation such as that following surgery, and thereby causing neural dysfunction manifesting as PNDs 9. The immediate effect of resistance exercise on peripheral and central inflammatory cytokines were analyzed using tissues (hippocampus, muscle and liver) obtained 30 min after the last resistance training session. Dynamic changes in the expression of inflammatory cytokines (TNF-α, IL-1β and IL-10) were demonstrated in the hippocampus (IL-10: 0.78 ± 0.22, p = 0.0048; Fig. 4a), muscle (IL-1β: 1.58 ± 0.61, p = 0.0276; IL-10: 6.7 ± 2.4, p = 0.0193; Fig. 4a) and liver (IL-1β: -0.42 ± 0.18, p = 0.0406; TNF-α: -0.59 ± 0.14, p = 0.0022; Fig. 4a). In conjunction with a reduction in pro-inflammatory cytokines in the liver, as well as the elevated levels of IL-10 in the hippocampus and muscle, resistance training induced an anti-inflammatory tendency both peripherally and in the CNS.
On postoperative day 1, we observed a significant increase in the mRNA expression of MCP-1 in the hippocampus of surgical mice compared to that of control (Interaction: F (1, 28) = 4.531, p = 0.0422; laparotomy factor: F (1, 28) = 5.756, p = 0.0233; exercise factor: F (1, 28) = 6.207, p = 0.0189. Multiple comparisons: 1.16 ± 0.36, p = 0.017, Figure 4b). Resistance exercise reduced the expression of MCP-1 in the hippocampus (-1.18 ± 0.36, p = 0.014, Figure 4b), no difference was seen in the expression of IL-1β,TNF-α and IL-10 (Fig. 4b).
3.5. Resistance exercise alleviates prolonged inflammatory response following surgery
The effect of resistance exercise on the sustained postoperative inflammatory response was examined on postoperative day 14. Microglia play a key role in the neuroinflammatory response in the brain and the number of Iba-1 label positive microglia was increased in the CA1 region of the hippocampus compared to control (interaction: F (1, 16) = 6.578, p = 0.0208; laparotomy factor: F (1, 16) = 12.57, p = 0.0027; exercise factor: F (1, 16) = 21.87, p = 0.0003. Multiple comparisons: 16.2 ± 3.8, p = 0.0032; Fig. 5a). Activated microglia with hypertrophic cell body were also present in the CA3 region of the hippocampus postoperatively compared to control (interaction: F (1, 16) = 3.053, p = 0.0997; laparotomy factor: F (1, 16) = 7.591, p = 0.0141; exercise factor: F (1, 16) = 18.23, p = 0.0006. Multiple comparisons: CA3: 0.33 ± 0.10, p = 0.0266, Fig. 5b). Preoperative resistance training attenuated this microglial activation by reducing their number (CA1: - 19.2 ± 3.8, p = 0.0005, Fig. 5a) as well as cell body size (CA3: -0.44 ± 0.10, p = 0.0031, Fig. 5b) compared to the laparotomy only group.
Astrocytes has been found to mediate microglial activation in surgery-induced neuroinflammation through MCP-1-CCR2 signaling 32. GFAP staining revealed a notable astrocyte proliferation in the DG region following surgery, with an increase in GFAP intensity (interaction: F (1, 12) = 3.753, p = 0.0766; laparotomy factor: F (1, 12) = 5.755, p = 0.0336; exercise factor: F (1, 12) = 6.664, p = 0.0240. Multiple comparisons: 0.84 ± 0.28, p = 0.043, Fig. 5e) and cell volume (interaction: F (1, 76) = 2.832, p = 0.0965; laparotomy factor: F (1, 76) = 9.053, p = 0.0036; exercise factor: F (1, 76) = 7.513, p = 0.0076. Multiple comparisons: 54.6 ± 16.5, p = 0.0075; Fig. 5h) compared to control. Preoperative resistance exercise ameliorated these changes compared to surgery alone (GFAP intensity: -0.88 ± 0.28, p = 0.034; cell volume: -51.58 ± 16.46, p = 0.013, Fig. 5d-h).
3.6. Alterations in myokines and Intracellular signal pathways following resistance exercise and surgery
Various forms of exercise, particularly of the aerobic type, have been shown to trigger production of myokines that can reduce neuroinflammation33 and confer positive effect in cognition34. PGC-1α is an important regulator of mitochondrial biogenesis, and it is known that Bcl-2 and Bax regulate the mitochondria-related intrinsic apoptosis by regulating the permeabilization of mitochondrial membrane, cytochrome C release and mitochondrial function35. The immediate effect of resistance exercise on myokines were analyzed by using tissues obtained 30 min after the last resistance training trail. The data showed that resistance training increased levels of myokines in the muscle (FGF-21: 5.38 ± 1.57, p = 0.0066; IL-6: 6.62 ± 1.49, p = 0.0012; Fig. 6a), liver (FGF21: 1.97 ± 0.90, p = 0.0537; PGC1-α: 0.788 ± 0.3371, p = 0.0415; IL-6: 1.141 ± 0.4779, p = 0.0381; Fig. 6b), and hippocampus (FGF-21: 2.58 ± 0.92, p = 0.0188; PGC1-α: 0.52 ± 0.22, p = 0.0405; Fig. 6c) as measured by RT-PCR.
The expression of BDNF/Akt/GSK3β and Bax/Bcl-2 were evaluated at 24 hours and 14 days after surgery, respectively. There was a decrease in BDNF (interaction: F (1, 20) = 3.59, p = 0.0727; laparotomy factor: F (1, 20) = 5.325, p = 0.0318; exercise factor: F (1, 20) = 19.31, p = 0.0003. Multiple comparisons: -0.408 ± 0.144, p = 0.035, Fig. 6e) and phospho-Akt at serine 473 (interaction: F (1, 20) = 9.614, p = 0.0056; laparotomy factor: F (1, 20) = 10.15, p = 0.0046; exercise factor: F (1, 20) = 3.137, p = 0.0918. Multiple comparisons: -0.463 ± 0.104, p = 0.0013, Fig. 6e) in the laparotomy group when compared to controls. Accordingly, surgery significantly increased the Bax/Bcl-2 ratio compared to control (interaction: F (1, 20) = 4.66, p = 0.0432, laparotomy factor: F (1, 20) = 18.44, p = 0.0004; exercise factor: F (1, 20) = 5.131, p = 0.0348. Multiple comparisons: -1.914 ± 0.42, p = 0.001, Fig. 6f) and prior resistance exercise attenuated these change (BDNF: 0.641 ± 0.144, p = 0.001; p-Akt: 0.359 ± 0.104, p = 0.0126, p-GSK3β: 0.6764 ± 0.1613, p = 0.0023; Bax/Bcl-2 ratio: -1.312 ± 0.42, p = 0.021, Fig. 6d-f) compared to the laparotomy group.
The changes in these markers were on day 14 similar except for phospho-Akt at serine 473 where no change was seen. The expression of BDNF (interaction: F (1, 20) = 4.673, p = 0.0429; F (1, 20) = 7.709, p = 0.0116; F (1, 20) = 4.937, p = 0.0380. Multiple comparisons: -0.6325 ± 0.1811, p = 0.0113, Fig. 6h) and phospho-GSK3β at serine 9 (interaction: F (1, 20) = 1.229, p = 0.2808; laparotomy factor: F (1, 20) = 9.424, p = 0.0060; exercise factor: F (1, 20) = 16.06, p = 0.0007. Multiple comparisons: 0.387 ± 0.131, p = 0.0362. Fig. 6h) was decreased. The Bax/Bcl-2 ratio was similarly increased (F (1, 20) = 15.83, p = 0.0007; laparotomy factor: F (1, 20) = 2.735, p = 0.1138; exercise factor: F (1, 20) = 11.5, p = 0.0029. Multiple comparisons: 0.774 ± 0.194, p = 0.004. Fig. 6i). Resistance exercise prior to surgery restored these changes to that comparable to the control (BDNF: 0.562 ± 0.181, p = 0.027; phospho-GSK3β at serine 9: 0.4739 ± 0.131, p = 0.0086; Bax/Bcl-2 ratio: 1.012 ± 0.1943, p = 0.0002, Fig. 6g-i).
3.7. Resistance exercise restored surgery-induced deficits in mitochondria density and morphology
Our data demonstrated a significant decrease in HSP60 in cytosolic fraction from the laparotomy group (interaction: F (1, 20) = 5.291, p = 0.0323; laparotomy factor: F (1, 20) = 5.194, p = 0.0338; exercise factor: F (1, 20) = 8.106, p = 0.010. Multiple comparisons: -0.5908 ± 0.1825, p = 0.0198, Fig. 7b). Importantly, a significant increase of cytochrome C was seen in the cytosolic fraction following surgery (interaction: F (1, 20) = 6.955, p = 0.0158; laparotomy factor: F (1, 20) = 18.45, p = 0.0004; exercise factor: F (1, 20) = 4.514, p = 0.0463. Multiple comparisons: 0.951 ± 0.194, p = 0.0005; Fig. 7c), both of which could be reduced by prior resistance exercise (RE + Lap compared to Lap, HSP60: -0.6641 ± 0.1825, p = 0.0082; cytochrome C: -0.6533 ± 0.194, p = 0.0149, Fig. 7b and c). There was no change in relative levels of HSP60 or cytochrome C in the mitochondria fractions (Fig. 7b and c).
Transmission electron microscopy (TEM) demonstrated a decreased in the total number of mitochondria after laparotomy (interaction: F (1, 172) = 7.266, p = 0.0077; laparotomy factor: F (1, 172) = 3.88, p = 0.0505; exercise factor: F (1, 172) = 8.03, p = 0.0052. Multiple comparisons: -0.3068 ± 0.9301, p = 0.0064; Fig. 7d and e), which was not seen in the RE + Lap group (RE + Lap compared to Lap: 0.3636 ± 0.9301, p = 0.0008; Fig. 7d and e). Although there was no significant difference between the control and surgery groups, resistance exercise significantly reduced the percentage of abnormal mitochondria (interaction: F (1, 172) = 3.039, p = 0.0831; laparotomy factor: F (1, 172) = 0.7192, p = 0.3976; exercise factor: F (1, 172) = 8.117, p = 0.0049. Multiple comparisons: -0.7502 ± 0.023%, p=0.0076; Fig. 7f-h).
3.8. Effect of resistance exercise and surgery on mitochondria dynamics.
Mitochondria undergo ‘mitochondrial dynamics’, which are critical for various cellular processes including inflammation, apoptosis and cell cycle, as well as mitochondrial quality control. We evaluated several important mitochondrial dynamic markers, including mitofusin-1, mitofusin-2, dynamin related protein 1 (Drp-1) and optic atrophy 1 (OPA1). Surgery-induced a significant increase of mitofusin-2 (interaction: F (1, 28) = 8.425, p = 0.0071; laparotomy factor: F (1, 28) = 14.28, p = 0.0008; exercise factor: F (1, 28) = 1.596, p = 0.2169. Multiple comparisons: 0.8668 ± 0.1835, p = 0.0004; Fig. 8b), and this upregulation was reduced by resistance exercise (-0.5405 ± 0.1835, p = 0.0379; Fig. 8b). No changes were demonstrated for the other markers. Mitofusin-2 is responsible for tethering endoplasmic reticulum to mitochondria (ER-mitochondria contacts) that allow Ca2+ influx from the ER to mitochondria and these were increased in the Lap (interaction: F (1, 76) = 9.632, p = 0.0027; laparotomy factor: F (1, 76) = 12.4, p = 0.0007; exercise factor: F (1, 76) = 14.4, p = 0.0003. Multiple comparisons: -0.7114 ± 0.1519, p<0.0001, Fig. 8c c-d) but not in the RE + Lap group (-0.7407 ± 0.1519, p<0.0001, Fig. 8c-d=.
Synapses are sites of high-energy demand that are particularly vulnerable to mitochondria dysfunction. Synaptosomes were isolated from the hippocampus and there was a significant increase of phospho-NR2A at tyrosine 1246 (interaction: F (1, 17) = 8.946, p = 0.0082; F (1, 17) = 12.49, p = 0.0026; F (1, 17) = 1.451, p = 0.2449. Multiple comparisons: -4.422 ± 0.9373, p = 0.0010; Fig. 8e and f) and phospho-NR2B at tyrosine 1472 (interaction: F (1, 17) = 6.592, p = 0.0200; laparotomy factor: F (1, 17) = 2.191, p = 0.1571; exercise factor: F (1, 17) = 3.088, p = 0.0969. Multiple comparisons: 1.547 ± 0.5286, p = 0.0424, Fig. 8e and f) following surgery. The level of phospho-AMPK-α at Thr172,which can be activated by excessive NMDA receptors activity, was enhanced following surgery (interaction: F (1, 17) = 6.955, p = 0.0173; laparotomy factor: F (1, 17) = 1.919, p = 0.1838, F (1, 17) = 4.573, p = 0.0473. Multiple comparisons: 0.4136 ± 0.1422, p = 0.0439; Fig. 8e and f). These laparotomy-induced changes were prevented by resistance exercise (p-NR2A: - 0.2843 ± 0.9373, p = 0.0343; p-NR2B: - 1.653 ± 0.5286, p = 0.0284; p-AMPK-α: - 0.491 ± 0.1422, p = 0.0146; Fig. 8e-g), indicating resistance exercise prevented NMDA receptor-mediated excitotoxicity.