Administration of monophosphoryl lipid A shortly after traumatic brain injury blocks the following spatial and avoidance memory loss and neuroinflammation

Abstract

Traumatic brain injury (TBI) frequently leads to cognitive impairments. The toll-like receptor 4 (TLR4) ligand, Monophosphoryl lipid A (MPL), has shown promise in modulating neuroinflammatory responses after TBI. We investigated the effects of MPL on spatial memory, passive avoidance memory, neuronal survival, and inflammatory/anti-inflammatory cytokines in rat brain following mild-to-moderate TBI. Rats underwent a learning period in the Morris water maze and shuttle box, followed by TBI induction by controlled cortical impact. MPL was administered into the cerebral ventricle 20 min after TBI. Spatial memory was assessed 7 and 28 days later. Passive avoidance memory was assessed 2 and 6 days after TBI. MPL significantly improved the spatial memory deficit at 7 days but not 28 days after TBI. It also improved impairment of the avoidance memory at both 2 and 6 days after TBI. MPL prohibited the TBI-induced TNF-α increase and IL-10 decrease in the injured region at 7 days post-TBI period. MPL prevented the neuronal loss induced by TBI in the hippocampus. A single administration of MPL shortly after TBI alleviates short-term memory deficits, through anti-inflammatory and anti-cell loss activities. Repeated MPL administration may also inhibit the long-term memory deficits after TBI.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-80331-3.

Traumatic brain injury (TBI) is a devastating condition that affects millions of people worldwide each year, leading to profound and debilitating consequences¹. Annually over 69 million individuals experience TBI², resulting in a cascade of complications, including emotional disturbances, cognitive deficit and chronic memory loss³. This global health crisis not only impacts patients’ quality of life but also places a significant burden on health care systems and society⁴. Memory loss is common across all TBI severity levels, disrupting daily life and impeding the ability to form new memories or recall past events^5,6.

The deficit in the spatial memory is a prevalent concern following TBI, impairing the ability of the injured person to navigate and remember locations. In the experimental studies investigating impact of TBI on the spatial memory, both learning development and memory retrieval are often assessed after TBI. However, assessing the retrieval of those memories which have been created before experiencing TBI, simulates clinical conditions more accurately. Subsequently, therapeutic interventions which are based on this approach, offer better translation to clinical practice.

Given the unpredictability of TBI, preventive strategies are clinically impractical. Instead, interventions applied after TBI hold greater potential for clinical applications. “Post-conditioning” is applying a sub-threshold noxious stimuli or a sub-effective dose of a ligand shortly after a major tissue damage, thereby promoting tissue resistance and protection against the damage⁷.

In the search to find drugs to prevent TBI neurologic consequences, Monophosphoryl lipid A (MPL) has shown promise⁸. MPL, is a semisynthetic derivative of lipopolysaccharide (LPS) that acts as a Toll-like receptor 4 (TLR4) agonist and stimulates the production of proinflammatory cytokines with the same potency as LPS but with one hundred times less toxicity⁹. MPL has been promising in immunotherapy of cancer, bacterial/viral infections, Alzheimer’s disease, and allergy^10,11. The excellent safety profile of MPL and good tolerance in humans, combined with high efficacy, underscores potential of this substance in treatment of a wide range of the inflammation-based diseases¹². Previous research has demonstrated that administering MPL can mitigate the adverse effects of the brain traumatic and ischemic injuries by anti-inflammatory effects. For example, MPL administration before or after TBI has inhibited neuroinflammation and the accelerated rate of seizures development in rats^8,13. MPL has recovered memory deficit when administered before ischemic brain injury¹⁴ or β-amyloid-induced neuroinflammation¹⁵. Also, MPL administration before induction of experimental epilepsy has demonstrated antiepileptic and anti-inflammation activities¹⁶. Moreover, MPL can reduce depressive-like behaviors in chronically stressed rats, via an anti-inflammation activity¹⁷.

Accordingly, the present study aimed to investigate the potential of MPL post-conditioning on averting the memory loss after TBI in rats, which have learned the relevant spatial and avoidance tasks before experiencing TBI. By administering MPL shortly after TBI, we assessed the memory in short- (2, 6, and 7 days) and long- (4 weeks) term post-TBI periods. Moreover, neuroinflammation state, and neuronal death in the main brain area involved in learning and memory, i.e., the hippocampus, was also assessed.

Results

Improvement in learning and memory during training

The 5-day Morris water maze training protocol resulted in significant improvement in the rats’ performance. Over the course of the training, the rats demonstrated a marked decrease in both the time and distance required to locate the hidden platform (Fig. 1A, B), indicating successful spatial learning and memory acquisition. Swimming velocity remained relatively constant throughout the assessment period (Fig. 1C).

Fig. 1.

Daily assessment of the spatial learning ability of rats during training in Morris water maze. (A) the latency, (B) the distance, and (C) the velocity of animals to reach the hidden platform. The animals’ learning improved over time, taking less time and swam a shorter distance to reach the platform. ****P < 0.0001. Velocity remained approximately constant across the 5 days of assessment.

Confirmation of the TBI severity

A deformation depth of 2 ± 0.02 mm was detected in the injured area (Fig. 2), indicating rats have experienced a mild-to-moderate TBI.

Fig. 2.

A coronal section of the rat brain with TBI, which is stained with TTC. It is taken at approximately 2 mm from bregma showing the extent of cortical damage after concussion by controlled cortical impact device. The dimensions of each square are 2 mm. An injury is seen in the right side with 2 mm deep from the cerebral cortex.

Improvement in the dysfunction of the TBI-induced spatial memory by MPL administration

The spatial memory was assessed in the probe phase by removing the platform and measuring the time spent in the target quadrant. At both the 7-day and the 28-day post-TBI periods, the latency to enter the target quadrant was significantly increased in the TBI + PBS group compared to the control and sham groups (P < 0.0001, Fig. 3A). MPL treatment reduced the latency in both 7-day and 28-day post-TBI periods [F (9, 66) = 1.0, P < 0.0001 and P < 0.05, respectively) indicating improved spatial memory retention.

Fig. 3.

Effect of MPL on the TBI-induced impairment in the spatial memory of rats. (A) the latency to first entry into the target quadrant, (B) the distance traveled in the target quadrant, (C) the time spent in the target quadrant. Data are shown as mean ± SEM (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D,E) Effect of mild-to-moderate TBI on the latency, and the speed to find the visible platform as visual-motor function of rats. No significant difference was observed among the groups.

The distance traveled to the target quadrant in the probe phase is demonstrated in Fig. 3B. At the both 7th and 28th days, the TBI + PBS group traveled more distance to find the platform compared to the control and sham groups (P < 0.01 and P < 0.05, respectively). A significant difference was observed between the TBI + MPL and TBI + PBS groups on the day 7 [F (9, 66) = 11.30, P < 0.01) but not on the day 28.

Figure 3C, shows the time spent by rats in the target quadrant. On the both 7th and 28th day post-TBI, the TBI + PBS group spent significantly less time in the target quadrant compared to the control, Sham + PBS, and Sham + MPL groups (F [9, 61] = 11.55, P < 0.0001, and P < 0.001, respectively). At the 7th but not the 28th day post-TBI period, the TBI + MPL group showed a significant increase in the time spent in the target quadrant compared to the TBI + PBS group (P < 0.001).

TBI did not change integrity of the motor and visual functions

The visible test confirmed that neither MPL nor TBI interfered with the visual-motor function of the animals. No significant difference was detected in the escape latency or swimming velocity among the groups (Fig. 3D, E). All rats were able to find the visible platform.

MPL inhibited impairment of passive avoidance memory by TBI

The extent of step-through latency in the experimental groups is demonstrated in Fig. 4. Rats with TBI showed significant lower latency at both 2 and 6 days after TBI compared to the other groups (P < 0.001). The injured rats which were treated by MPL showed significant different latency compared to the TBI group. MPL itself does not affect the latency compared to the sham group.

Fig. 4.

Effect of TBI and MPL on the passive avoidance memory of rats. Data are shown as mean ± SEM (n = 8). * and ^#P < 0.001 compared to the trauma group.

MPL prevented the TNF-α increase by TBI at 7 days post-TBI period

The results of TNF-α assay by western blot (WB) and ELISA techniques are demonstrated in Fig. 5. Injection of PBS to sham-operated animals (Sham + PBS group) did not significantly change TNF-α level at the both 7 and 28 days after injection, compared to control group. Also, injection of MPL to sham-operated animals (Sham + MPL group) did not change TNF-α level compared to Sham + PBS group at the both 7 and 28 days after injection. TNF-α showed significant increase at both 7 and 28 days after TBI (TBI + PBS group), compared to Sham + PBS group (WB: P < 0.05, and P < 0.001; ELISA: P < 0.0001 for both time periods). At 7 days post-injection period, the TNF-α level in TBI + MPL group was significantly lower than TBI + PBS group (WB: P < 0.01; ELISA: P < 0.0001), and not significantly different from Sham + MPL, and Sham + PBS groups (P > 0.05). At the 28 days period, the TNF-α level in TBI + MPL groups had no statistically significant difference with TBI + PBS group.

Fig. 5.

Effect of MPL on the TNF-α level in the TBI area. (A) The representative blots of the rat brain TNF-α and β-actin (internal control) obtained by western blot 7 and 28 days after TBI or sham operation. (B) The mean ratio of TNF-α to ß-actin in the experimental groups. (C) TNF-α level in the TBI region obtained by ELISA technique. Data are represented as Mean ± SEM of three repeats each representing the mean value of pooled samples of six rats per group, which analyzed by Kruskal–Wallis test and Dunn’s supplementary test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0001.

MPL inhibited the IL-10 decrease by TBI the 7 days post-TBI period

The results of IL-10 assay by western blot and ELISA techniques are demonstrated in Fig. 6. Injection of PBS to sham-operated animals (Sham + PBS group) did not significantly change IL-10 level at the both 7 and 28 days after injection, compared to control group. Likewise, injection of MPL to sham-operated animals (Sham + MPL group) did not change IL-10 level compared to Sham + PBS group at the both 7 and 28 days after injection. IL-10 was significant decreased at both 7 and 28 days after TBI (TBI + PBS group), compared to Sham + PBS group (WB: P < 0.01, and P < 0.05; ELISA: P < 0.0001 for both time periods). The IL-10 level in TBI + MPL group was significantly different from TBI + PBS group (WB: P < 0.01; ELISA: P < 0.0001) at 7 days post-TBI period. No significant difference was found in IL-10 level between TBI + MPL and Sham (Sham + MPL, and Sham + PBS) groups at 7 post-injection period (P > 0.05). At the 28 days period, no statistically significant difference was detected between TBI + MPL and TBI + PBS groups.

Fig. 6.

Effect of MPL on the IL-10 level in the TBI area. (A) The representative blots of the rat brain IL-10 and β-actin (internal control) obtained by western blot 7 and 28 days after TBI or sham operation. (B) The mean ratio of IL-10 to ß-actin in the experimental groups. (C) IL-10 level in the TBI region obtained by ELISA technique. Data are represented as Mean ± SEM of three repeats each representing the mean value of pooled samples of 6 rats per group, which analyzed by Kruskal-Wallis test and Dunn’s supplementary test. ** and ****, P < 0.05 and P < 0.0005, respectively). *P < 0.05, **P < 0.01, and ****P < 0001.

MPL suppressed TBI-induced cell damage in CA1 and CA3 hippocampal sub-regions

Morphometric alterations in hippocampal areas including CA1, CA3, and dentate gyrus (DG) of the experimental groups at 7 days after TBI or sham operation are presented in Fig. 7. The shrunken neurons with pyknotic nucleus in CA1 and CA3, were significantly high in TBI + PBS group (P < 0.0001), compared to Sham + PBS group (Fig. 7F and G). No significant change was observed in DG of TBI + PBS group compared to Sham + PBS (Fig. 7H).

Fig. 7.

Effect of post-TBI injection of MPL on the TBI-induced neuronal loss in the rat hippocampus 7 days after TBI. (A) a schematic coronal section of the rat brain adapted from Paxinos and Watson (2007) rat brain atlas that closely resembles brain areas demonstrated in B-E. (B–D) The sample images of the Nissl-stained rat brain sections showing CA1, CA3 and dentate gyrus (DG). (E) The sample images of the Nissl-stained rat brain sections of the experimental groups showing CA1, CA3, and DG with higher magnification. (F–H) Number of the living neurons in CA1, CA3, and DG areas in the experimental groups. Each histogram represents mean ± SEM of the number of undamaged neurons in the side of the brain that is underwent TBI. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0001. Scale bar = 100 μm.

As seen in Fig. 7, injection of MPL to sham-operated animals (Sham + MPL group) did not change number of living neurons in the hippocampal sub-regions at 7 days post-injection period. However, by injection of MPL to rats with TBI (TBI + MPL group), the damaged or necrotic neurons in the CA1 and CA3 regions of the hippocampus was significantly lower than those in the TBI + PBS group (P < 0.05, and P < 0.01, respectively), and not significantly different from Sham + MPL, and Sham + PBS groups (P > 0.05). No significant change was observed in DG of TBI + MPL group compared to TBI + PBS (Fig. 7H).

Discussion

In this study, we examined effects of mild-to-moderate TBI in the temporo-parietal cortex, on the spatial and passive avoidance memories after TBI. At the all post-TBI periods (2, 6, 7, and 28 days) the rats were unable to retrieve the pre-injury established memory. TBI induced imbalance in the cytokines TNF-α and IL-10 toward neuroinflammation. Moreover, TBI was accompanied by significant neuronal cell death in the CA1, and CA3 region of the hippocampus. Injection of MPL 20 min after TBI significantly prevented the impairment of the spatial and passive avoidance memories observed at the 2, 6, and 7 days post-TBI periods. Furthermore, MPL adjusted the imbalance of TNF-α and IL-10, and prevented the death of hippocampal CA1 and CA3 neurons in the one-week post-TBI period. At the 28-days post-TBI period, MPL could partly improve the memory deficit but could not affect the cytokine imbalance and hippocampal cell death.

CCI, as an animal model of TBI, induces focused brain injury with pathobiological changes similar to clinical head injuries¹⁸. In the present study we showed that CCI injury leads to permanent impairment of spatial memory in 1 and 4 weeks after TBI. This finding is consistent with previous studies indicating that TBI can cause persistent cognitive deficits. It is reported that the spatial memory is impaired in rats, beginning 2 weeks after CCI and continued for 12 months thereafter¹⁹. In another study, CCI-injured rats showed impaired spatial memory after 4 months along with disruption of the fear memory and novel object recognition²⁰. In the present study, CCI injury also impaired passive avoidance memory in 2 and 6 days after TBI. Effect of TBI on the passive avoidance memory has been examined previously in different experimental models of TBI. For instance, it has been shown that mild TBI can negatively affect the avoidance memory of mice 4 weeks after CCI²¹. However, 10 days after fluid percussion injury, retention of passive avoidance memory has not been affected in rats²². Regardless of whether the memory is influenced by the concussion or not, in all previous studies, the training and consolidation of the memory has been performed after concussion. However, in our study, learning process and memory acquisition is performed before TBI. Then the memory retention was assessed at the short and long term periods after TBI. In terms of the deterioration of those memories which were formed before TBI, our approach has more translability to clinical situations.

TLRs are in the frontline of the innate immune system that switches on immune responses. Microglia, the only innate immune cells in the central nervous system, highly express TLRs particularly TLR4²³. TLR4 is up-regulated following TBI and contributes to TBI pathology. Therefore inhibition of TLR4 by pharmacologic^24–28 and genetic tools^29–31 reduces TBI neurological complications. Nonetheless, it is shown that TLR4 priming by the TLR4 agonist MPL, effectively prevents the β-amyloid-induced memory deficit and overproduction of proinflammatory cytokines¹⁵. Moreover, TLR4 preconditioning by MPL, 2 days before hippocampal ischemia reduces infarct size, improves cognitive function, down-regulates inflammatory mediators, NF-κB and TNF-α, and upregulates anti-inflammatory mediators, IRF3, IFN-β, and TGF-β¹⁴. In the current study, we found that administration of TLR4 ligand MPL, shortly after TBI inhibits the one-week post-TBI cognitive deficit. However, the single post-TBI administration of MPL, could not inhibit the destructive effect of TBI on the four-week post-TBI cognitive function. It seems that the single dose administration of MPL is effective for 1 week after injection, and for longer duration of action, the repeated administration is needed.

TNF-α plays a crucial role in TBI, affecting both the onset of the acute phase and the progression of injuries³². TNF-α regulates synaptic plasticity and long-term potentiation³³. However, excessive TNF-α disrupts synaptic plasticity and causes neuronal damage, leading to memory loss^32,34,35. After TBI, elevated TNF-α levels in the damaged brain areas trigger inflammation and secondary brain damage³². Studies indicate that inhibiting TNF-α after TBI can reduce inflammation and aid in memory recovery^34,35. Therefore, a part of beneficial effect of MPL on the TBI memory destruction might be mediated through anti-inflammatory activity by preventing TNF-α production after TBI. To address the role of TNF-α in the improving the effect of MPL on cognitive deficits after TBI, we measured TNF-α level in the contused brain region at 1 and 4 weeks post-TBI- periods. We found a significant increase in TNF-α level at both time periods. The previous study have shown that TNF-α protein significantly increases in the rat brain at 3, 6, 12 h³⁶ 24 h³⁷, 1 week^38,39 and 30 days³⁹ after TBI. In our study, injection of MPL immediately after TBI prevented the surge of TNF-α in 1 week but not 4 weeks post-TBI period. Previous researches have demonstrated that MPL can improve acute neuroinflammation and cognitive function within 24 h after injury^14,15. Our findings suggest longer duration of action of MPL than 1 day, as it persisted till 7 days after TBI. Since we applied a single administration of MPL, the one-week preventive effect of MPL on the TNF-α rise and the memory impairment, suggests a one-week biological activity for the single administration MPL. In line with our study, it is shown that i.p. administration of 50 µg MPL to mice with the invasive pneumonia induces cytokine response including TNF-α surge in serum for a period between 2 and 12 h. However, the survival rate of the infected mice at day 7 increased from 0% in untreated animals to 50% in MPL-treated mice. The authors suggested that a combination of TLR4 priming and the time scale of MPL peak biological activity is responsible for this one-week action duration of MPL⁴⁰.

IL-10 as an anti-inflammatory cytokine has a critical role in the response to TBI. It inhibits the production of inflammatory cytokines such as TNF-α and IL-6, helping to maintain synaptic plasticity and neuronal function, which are essential for learning and memory processes⁴¹. IL-10 plays a crucial role in memory by inhibiting inflammation and supporting neuronal survival and function. Elevated levels of IL-10 after TBI have been shown to reduce inflammation and improve cognitive outcomes⁴¹. Our observations show a significant decrease in IL-10 at day 7 after TBI. Similarly, it is shown that TBI in rats is accompanied by IL-10 decrease at 24 and 48 h after CCI^13,36. On the other hand, an increase in expression of IL-10 has been reported in rats experiencing TBI, from 1 day until 4 weeks after CCI^39,42. In our study, MPL resets IL-10 concentration in the TBI state to the control level. Similar to our study, MPL has increased the dropped level of IL-10 by CCI or β–amyloid neuroinflammation to the control level^13,15.

We have recently shown that MPL administration, by the same dosing and timing as the present study, suppresses TNF-α increase, enhances IL-10 expression, and polarizes microglia M1 inflammatory phenotype to the M2 anti-inflammatory phenotype in rats with TBI¹³. Therefore, polarization of microglia to anti-inflammatory state have been involved in the preventive effect of MPL on impairment of memory after TBI.

In our study, TBI was accompanied by significant neuronal death in CA1 and CA3 regions of hippocampus. Given the central role of the hippocampus in the memory and especially spatial memory⁴³, the CA1 and CA3 cell death can contribute to the destructive effect of TBI on spatial memory. In our study, TLR4 ligand MPL could reduce neuronal death in the hippocampus. TLR4 has shown both beneficial and harmful effects against TBI neurodegeneration. The TLR4 genetic ablation³¹, and/or pharmacologic inhibition^24,27,44, have inhibited brain injury and cell death induced by TBI. On the other hand, preconditioning of TLR4 by the TLR4 agonists including LPS or MPL has reduced injury volume and cell death of TBI^45,46, and radiation⁴⁷. Moreover, TLR4 post-conditioning by LPS or MPL have been protective against ischemia- or CCI-induced cell loss^13,48. Our results are in line with these studies supporting protective action of the TLR4 agonist MPL against TBI neuronal damage.

Although MPL could slightly improve the memory deficit 28 days after TBI, this effect was not statistically significant. It seems that a single administration of MPL has not been sufficient for long-term protective activity against TBI. In line with this proposal, it has been reported that repeated MPL injections every 10 days, and not a single injection, is necessary to maintain consistent anti-depressive effects in a rodent model of stress-induced depression⁴⁹.

In conclusion, our study disclosed another experimental evidence on the beneficial effect of TLR4 post-conditioning in preventing TBI adverse effects, particularly memory loss in the focused brain injury. Further studies are necessary to confirm the optimal dosing regimen of MPL for long-term cognitive recovery after TBI. Moreover, assessing effectiveness of MPL in other types of TBI, such as diffuse TBI, and blast-induced neurotrauma renders our study applicable to a wide range of TBI.

Limitations of the study

We tested the effect of MPL on TBI memory deficit only in male rats. Including the female sex in the future studies will add the comprehensiveness of the study. In our study, MPL single administration was effective till 1 week after injection. The repeated administration of MPL might have longer beneficial effects and prevent the 28 days post-TBI memory deficit. In addition, we administered MPL into cerebral ventricles. Evaluating the effect of systemic administration of MPL on the TBI adverse effects will increase translation of the study to clinical practice.

Methods

Animals

Adult male Wistar rats (250–270 g, 2.5 months old) were obtained from Pasteur Institute of Iran (n = 120). They were housed in groups of four in polypropylene cages with ad libitum access to food and water under a 12-h light/dark cycle (08:00–20:00) at a controlled temperature of 23 ± 1 °C. All animal procedures were approved by the Review Board and Ethics Committee of Pasteur Institute of Iran (Authorization code IR.PII.REC.1400.087) and were performed in accordance with the ARRIVE guidelines and regulations for the use of animals for scientific purposes by Council Directive 2010/63EU of the European Parliament, and the Council of 22 September 2010.

The sample size was calculated by extrapolation of outcomes from our previous study¹⁵. Given to the expected effect size 2.6, type I error of 0.05, power of 0.8, an allocation ratio of 1:1, the raw sample size per group was obtained 7 using a free access software (https://www.statskingdom.com/sample_size_regression.html). With an attrition of 10% (possibility of death after traumatic brain injury), the corrected sample size per group was obtained 8 (7/0.9 = 7.77). Given to allocation of 15 experimental groups, 120 rats were used totally.

Induction of mild to moderate TBI and MPL administration

General anesthesia was achieved by intraperitoneal (i.p.) co-injection of ketamine (100 mg/kg; Alfasan, The Netherlands) and xylazine (10 mg/kg; Alfasan, The Netherlands). After confirming deep anesthesia, the coordinates of the left lateral ventricle of the rat brain were determined using the Paxinos and Watson atlas⁵⁰ and marked on the skull surface at AP: −0.96, and ML: −1.8. The marked area was drilled using a dental drill under sterile conditions. Next, a 5 mm diameter burr hole was drilled at right side of the skull bone, with coordinates 4 mm posterior and 4 mm right of the bregma. The bone was removed and the exposed parietotemporal cortex was injured using a Controlled Cortical Impact (CCI) impactor (AmScience Instruments, USA) equipped with a 5 mm diameter piston. In order to create a mild to moderate TBI with cortical deformation depth of 2 mm, the device was set to the impactor velocity of 4.5 m/sec, and 150 msec duration of the impact⁵¹.

Immediately after trauma induction, the circular piece of the skull was repositioned and secured with dental cement. After 20 min, 1 µg/5µl MPL (Sigma-Aldrich Co., USA) was injected into the lateral ventricle of the brain (i.c.v.) at the pre-marked area, 3.4 mm deep from the dura surface towards the brain interior using a 30-gauge needle attached to a 5-µl Hamilton syringe by a polyethylene tube. The dosage was selected based on our previous studies^8,13. MPL was dissolved in the sterile phosphate buffer solution (PBS) with a pH range of 7.5. Finally, the skull surface was disinfected with betadine, and the scalp is sutured. The rats were transferred to a warm location to recover. Sham-operated animals underwent the whole TBI procedure except that they did not receive CCI injury.

Morris water maze

The Morris water maze test was used for spatial learning and memory as our previous study¹⁵. The learning protocol consisted of a 5-day spatial acquisition phase followed by a 1-day probe trial. The maze featured a black pool with a diameter of 150 cm and a height of 50 cm, filled with water maintained at 23 ± 2 °C to a depth of 25–30 cm. A 10-cm-diameter platform was submerged 2 cm below the water surface in a designated quadrant. During the acquisition phase, rats underwent four trials per day for five consecutive days, with an inter-trial interval of 10 min. The rats were released from various starting points and tasked with locating the hidden platform, with successful navigation indicating effective spatial learning. Memory retention was assessed using the spatial probe test on the 7th and 28th day post-surgery. In the probe trial, the platform was removed, and the rats were placed in the quadrant opposite to the previous platform location for a 1-min exploration period. Key metrics recorded included the latency to first entry to target quadrant, distance traveled to the former platform location, and the time spent in the target quadrant. Data were analyzed using EthoVision XT 7 software (The Netherlands) and manually scored by an experimenter blinded to the experimental conditions.

Shuttle box

The shuttle box apparatus was used to measure passive avoidance memory of the animals, according to our previous study⁵². The apparatus consisted of two chambers, bright and dark (20 cm × 20 cm× 30 cm), which are separated by a guillotine door (7 cm × 9 cm). Bottom of the chambers are made of stainless steel grids with 2.5 mm diameter and 1 cm intervals. The bottom of the dark compartment is connected to an insulated stimulator. At the first day (Day−1), the rat was placed in the bright chamber and guillotine door was opened allowing the animal to explore the apparatus for 60 sec. The animal was then returned to its cage. Thirty min later, the procedure was repeated as training session and a foot shock (50 Hz, 1 mA for 5 sec) was applied once the animal entered the dark chamber. After 20 sec, the animal was returned to its cage. Two min later, the procedure was repeated and the acquisition was attained if the rat would not enter the dark chamber for 300 sec as the step-through latency. Twenty-four h later (Day 0), the rat was placed in the light chamber, the door was opened and its behavior was monitored for 10 min and the step-through latency was recorded. Then, the rats were underwent TBI, and step-through latency was measured 2 and 6 days after TBI.

Western blot

By completing the probe test of spatial memory, rats were sacrificed for western blot analysis. Tissue samples were homogenized with RIPA lysis buffer for western blot analysis. The homogenates were centrifuged at 14,000 rpm for 20 min at 4 °C, and the supernatant protein concentration was determined using the Bradford Protein Quantification kit (DB0017, DNAbioTech, Iran) according to the manufacturer’s instructions. Equal volumes of tissue lysates and 2× Laemmli sample buffer were mixed. The lysates (20 µg) were boiled for 5 min at 95 °C, then subjected to SDS-PAGE on a 12% gel and transferred to a 0.2 μm PVDF membrane (Immune-Blot™, Bio-Rad Laboratories, CA, USA). Membranes were blocked with 5% BSA (Cat No: A-7888; Sigma Aldrich, MO, USA) in 0.1% Tween 20 for 1 h at room temperature, then incubated with primary antibodies for 1 h at room temperature. The used primary antibodies included: Rabbit polyclonal anti-TNF-α antibody (17, 26 kDa; 1:1000 dilution; Cat no ab307164; Abcam), Rabbit polyclonal anti-IL-10 antibody (20 kDa; 1 µg/ml dilution; Cat no ab9969; Abcam), Rabbit monoclonal anti-β-actin antibody (42 kDa; 1:5000 dilution; Cat no ab8227; Abcam). After three washes with TBST, membranes were incubated with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1:10,000, Cat No: ab6721; Abcam) for 2 h at room temperature. The membranes were washed six times for 10 min each in TBST and incubated with enhanced chemiluminescence (ECL) for 1–2 min. Protein bands were visualized in the dark room, and exposed to autoradiography film (Kodak, Shenzhen, China). The density of bands was measured by the ImageJ 8.0 software. The density of each band was divided by the density of its corresponding β-actin band, and the calculated values were compared between groups.

ELISA

IL-10 and TNF-α were measured in the protein homogenates by quantitative sandwich enzyme immunoassay using Abcam antibodies (ab9969 for IL-10, and ab307164 for TNF-α). The procedure was performed according to the manufacturer’s instructions.

Histology

Measurements of lesion size

The size of TBI was evaluated by 2, 3, 5- triphenyl tetrazolium chloride (TTC; Sigma-Aldrich, Canada) staining. The rats were euthanized by CO₂ 95%, and decapitated. Brains were removed and cut freshly into 2 mm thick coronal slices. The slices were incubated in 0.9% NaCl containing 2% TTC at 37 °C for 15 min. The unstained area was considered as damaged area. The depth of injured tissue was manually quantified by a micrometer.

Nissl staining

At the end of experiments, rats were deeply anesthetized, and immediately perfused with 4% paraformaldehyde (Merck, Germany) in 0.1 M PBS, through the ascending aorta. The brains were collected and kept in a 4% formaldehyde solution overnight at 4 °C. The fixed tissues were then dehydrated, vitrified, and embedded in paraffin. Sections of 5 μm thickness were prepared. Then from beginning of the injured region, out of every 10 sections, the 9th section was selected for staining. The brain sections were dewaxed, rehydrated, and microwaved in 0.01 M sodium citrate buffer for 5 min. After cooling to room temperature and washing with PBS three times, the sections were stained with cresyl violet ((Merck, Germany) 0.5%. The stained sections were then dehydrated with 95% ethanol for 5 min, followed by 100% ethanol for 10 min, and finally cleared in xylene for 10 min. The sections were mounted and visualized using an optical microscope (Olympus Optical Co. Ltd., Japan) at 40× magnification.

Experimental design

Eighty rats were subjected to learning process (acquisition of the spatial task) and underwent a training phase in the Morris water maze for 5 days. After formation of the spatial memory, animals were randomly divided into 10 groups of eight rats in each. There were two control groups including the 7-day control group (memory was checked on the 7th day after learning the water maze), and the 28-day control group (memory was assessed on the 28th day after learning phase). There were 2 Sham + PBS groups including the 7-day, and the 28-day groups in which the rats received PBS i.c.v. 20 min after sham operation, and memory was checked on the 7th or the 28th day post-injection periods. There were 2 Sham + MPL groups including the 7-day, and the 28-day groups. The rats underwent similar procedure as the Sham + PBS groups except for receiving MPL instead of PBS. There were two TBI + PBS groups including the 7-day and the 28-day groups. The rats underwent similar procedure as the Sham + PBS groups except for experiencing TBI instead of sham operation. There were 2 TBI + MPL groups including the 7-day, and the 28-day groups. The rats underwent similar procedure as the TBI + PBS groups except for receiving MPL instead of PBS.

After evaluating memory using the Morris water maze probe test, histological (using two rats from each group) and cytokine assays by western blot and ELISA (using six rats from each group) were performed.

The other 40 rats were allocated for passive avoidance test. After consolidation of the avoidance memory, the animals were randomly divided into five groups (eight rats in each group) including control, Sham + PBS, Sham + MPL, TBI + PBS, and TBI + MPL, and treated with the same procedure as the experimental groups described for the spatial memory assessment. The avoidance memory of the rats was then assessed 2 and 6 days after TBI or sham operation.

A flowchart illustrating the experimental design and timeline of the study is demonstrated in Fig. 8.

Fig. 8.

Outline of the study design and experimental procedures. Eighty rats, underwent Morris water maze learning for four sessions every day for 5 days. In the fifth day, the TBI induction performed by CCI. Forty rats were trained on the one-trial passive avoidance task with retention assessed 2 and 6 days after TBI. MPL/PBS injected into cerebral ventricles 20 min post-injury. The TNF-α, and IL-10 levels was assessed by western blot and ELISA. The neurodegeneration was analyzed by Nissl staining. All experiments were done 7 and 28 days after TBI.

Statistical analysis

The collected data were expressed as mean ± SEM and analyzed using GraphPad Prism 9.4 software. The normality of data distribution was assessed using the Shapiro-Wilk test, confirming normal distribution (P > 0.05). Levels of cytokines and protein density relative to β-actin were evaluated using non-parametric Kruskal–Wallis test followed by Dunn’s post hoc correction. Metrics from the Morris water maze, including time spent in the target quadrant, escape latency, and distance to the platform, were analyzed. Time spent in the target quadrant, the step-through latency of the shuttle box test, and the mean number of necrotic neurons were analyzed using one-way ANOVA with Dunnett’s post hoc correction. Escape latency and distance to the platform were analyzed using two-way ANOVA with Bonferroni post hoc test. Statistical significance was set at P < 0.05 for all tests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Study design: MS, HGH. Data acquisition and analysis, drafting of figures and manuscript: MH, MRS, MKM. Interpretation of results: MS, HGH. Editing and finalizing manuscript: MS, AA.

Data availability

All data are available from corresponding author (sayyahm2@pasteur.ac.ir) upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.