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Minocycline attenuates neurological impairment and regulates iron metabolism in a rat model of traumatic brain injury
Archives of Biochemistry and Biophysics 682 (2020) 108302
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Minocycline attenuates neurological impairment and regulates iron metabolism in a rat model of traumatic brain injury
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Lijun Zhanga, Hong Xiaob, Xing Yub, Yongbing Dengb,∗ a b
Department of Neurosurgery, First People's Hospital of Taizhou, Taizhou, Zhejiang, 318020, China Department of Neurosurgery, The Fourth People's Hospital of Chongqing, 400014, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Traumatic brain injury Minocycline Neurological impairment Iron metabolism
There is currently no effective treatment for neurological impairment caused by traumatic brain injury (TBI). It has been reported that excessive iron production in the brain may be a key factor in neurological impairment. In the present study, we investigated the effects of minocycline, a semi-synthetic tetracycline antibiotic, against TBI-induced neurological impairment and explored its underlying mechanism. Neurological impairment was assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Nissl staining was performed to evaluate cell viability in the brain. The iron concentrations in cerebrospinal fluid (CSF), serum, and brain tissues were examined. The Fe2+- and Fe3+- chelating activity of minocycline was measured. Finally, the expression levels of important iron metabolism proteins ferritin, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hepcidin in the hippocampus and cortex were measured by Western blot analysis. The results indicate that minocycline significantly attenuated the neurological impairment caused by TBI and increased neuronal viability. Minocycline showed a Fe2+- and Fe3+- chelating activity in vitro and reduced the iron concentration in CSF and brain tissues (cortex and hippocampus). Minocycline also inhibited the overexpression of ferritin and TfR1, but did not affect the expression of DMT1. Minocycline restored the expression of FPN1 by decreasing the expression of hepcidin. In conclusion, minocycline may attenuate neurological impairment caused by TBI and regulate iron metabolism.
1. Introduction Traumatic brain injury (TBI) is a major cause of trauma, which is responsible for the majority of death in individuals aged 1–45 [1]. According to a survey conducted in 2016, the rates of emergency department visits, hospitalizations, and deaths related to TBI have significantly increased from 2001 to 2010 [2]. The incidence of TBI has rapidly increased due to traffic accidents, wars, accidents, and natural disasters [3]. Approximately 60% of TBI cases and 70% of severe head trauma cases are caused by car accidents in China [4]. With the development of modern medical technology, the mortality rate of TBI patients has significantly decreased, but many patients have long-term motor, sensory, behavioral, and cognitive dysfunction [3]. For secondary injuries caused by TBI, such as axonal rupture, neuronal apoptosis, brain atrophy, and glial scar formation, and subsequent behavioral and cognitive dysfunction, no effective treatment is yet available. At present, the pathological mechanisms involved in secondary injury of TBI include neuronal apoptosis, inflammation, oxidative
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damage, and disorders of energy metabolism [5–7]. Many studies have shown that overproduction of iron in the brain has a neurotoxic effect and is involved in neurodegenerative diseases and intracerebral hemorrhage [8,9]. Studies have reported that intracerebroventricular injection of autologous blood and FeCl3 induced chronic hydrocephalus, whereas treatment with an iron-chelating agent (deferoxamine; DFO) reduced the incidence and severity of hydrocephalus and improved Morris water maze test results [10,11]. These results suggest that excessive iron in the ventricle has a neurotoxic effect, and DFO can attenuate the neurotoxicity by reducing excessive iron. Studies of intracerebral hemorrhage [12,13] and neurodegenerative disease [14] revealed that excessive iron may be essential in brain atrophy and neurological impairment. An increase in brain iron content was observed 1 day after experimental intracerebral hemorrhage and lasted for 3 months [12]. Xi et al. found that injecting Fe3+ into brain parenchyma can cause brain parenchymal injury [13]. Minocycline, a semi-synthetic tetracycline antibiotic, has good lipid solubility and can easily pass through the blood–brain barrier. Experimental studies have found that minocycline can protect neurons
Corresponding author. Department of Neurosurgery, The Fourth People's Hospital of Chongqing, Chongqing, 400014, China. E-mail address: [email protected] (Y. Deng).
https://doi.org/10.1016/j.abb.2020.108302 Received 18 September 2019; Received in revised form 8 February 2020; Accepted 8 February 2020 Available online 10 February 2020 0003-9861/ © 2020 Published by Elsevier Inc.
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similar to TBI. Rats in the TBI + Vehicle group received TBI treatment and intraperitoneal (i.p.) saline injection. Rats in the TBI + MINO-10, TBI + MINO-20, TBI + MINO-40 group received TBI treatment and i.p. injection of 10 mg/kg, 20 mg/kg, and 40 mg/kg of minocycline. Rats in the TBI + Deferiprone group received TBI treatment and i.p. injection of deferiprone (50 mg/kg, Apopharma, Inc., Canada). Rats in the MINO20 group received i.p. injection of 20 mg/kg minocycline only. Daily injection of minocycline (Sigma, St. Louis, MO, USA) started at 12 h post-injury and continued for 7 days. Rat body weights were measured daily post-injury. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition (National Academies Press, Washington, DC, 2010).
against intracerebral hemorrhage, ischemia, brain trauma, and Alzheimer's disease [15–19]. It is generally believed that its effects include inhibition of microglia activation and inflammation through its anti-oxidant and anti-apoptotic actions [20–22]. Minocycline is also reported to inhibit metalloproteases, inducible nitric oxide synthase, and poly (ADP-ribose) polymerase [23,24], but the exact mechanism is not yet clear. On the other hand, although there are many studies that have found beneficial effects of minocycline treatment following TBI, there are also mixed results, with some studies showing minimal or no beneficial effects. For example, Sanuki et al. found that minocycline did not increase the survival of aged penetrating TBI flies [25]. Scott revealed that minocycline reduced chronic microglia activation but increased neurodegeneration in a TBI model [26]. Therefore, it remains significative to examine the effect of minocycline on different markers of TBI. In brain tissue, most iron ions bind to proteins in the form of stable ferritin. Ferritin consists of two different homologous proteins, ferritin light chain (FL) and ferritin heavy chain (FH). Transferrin (Tf) is a single chain polypeptide with a molecular weight of 75 kg/mol. First, iron ions in the cerebrospinal fluid (CSF)/brain interstitial fluid bind to Tf and form a complex with transferrin receptor (TfR). The complex forms a phagocytic vacuole and enters the cell, in which Fe3+ reductase deoxidizes Fe3+ to Fe2+, which is then transported to the cytoplasm and participates in cellular functions. After the release of iron ions, the Tf/TfR complex returns to the cell surface, where Tf is released to the extracellular matrix. TfR is mainly located in oligodendrocytes, suggesting that these cells take in ferric ion mainly through the Tf/TfR pathway. The main function of divalent metal transporter 1 (DMT1) is to mediate the entry of Fe2+ into the cell and transport Fe2+ from the endocytic vesicles into the cytoplasm. A G185R mutation in the DMT1 gene may cause severe iron deficiency anemia in mice [27]. DMT1 is closely related to human diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease [28]. Ferroportin 1 (FPN1), also known as iron-regulated transporter 1 (IREG1) or metal transporter protein 1 (MTP1), is a transmembrane iron export protein. The main function of the FPN1 protein is to transport iron and manganese in the cell into the plasma, releasing iron from the cell [29]. FPN1 is highly expressed in rat hippocampus, cerebral cortex, cerebellum, thalamus, and striatum. Hepcidin acts as an important protein regulating the expression of FPN1 [29]. Whether and how minocycline regulates these proteins attracted our curiosity. In the present study, we used a rat model of TBI, a weight drop model, to explore the role of iron metabolism in the neuroprotective action of minocycline. The effect of minocycline on neurological impairment was assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Nissl staining was performed to evaluate cell viability in the brain tissue. The Fe2+- and Fe3+- chelating activity of minocycline was measured. The iron concentrations in CSF, serum, and brain tissues were examined. Finally, several important iron-related proteins, ferritin, TfR1, DMT1, FPN1, and hepcidin, were measured by Western blot analysis to explore the effect of minocycline on iron metabolism.
2.2. Rat model of focal TBI The TBI procedure, a weight drop model, was similar to that described by Li et al. [30]. First, rats were given ketamine (90 mg/kg, ip) and xylazine (10 mg/kg, ip) for complete anesthesia. Next, the skull was exposed after a midline scalp incision. A left parietal bone window was made using a dental drill (+3.0 mm posterior; 2.7 mm lateral to the bregma). Trauma was inflicted by a cylindrical steel rod (diameter, 4 mm; weight, 40 g) that fell from a height of 25 cm onto a piston on the surface of the dura mater. Finally, the incision was closed and lidocaine jelly was used to prevent infection. Rats in the Sham group were subjected to the same procedure, except the cylindrical steel rod did not fall. H&E staining of the cortex of 3.0 mm posterior to the bregma in the injured and contralateral hemispheres was performed to show the impact site. 2.3. Assessment of neurological impairment At 7 days post-injury, neurological impairment was assessed once daily by foot-fault test, cylinder test, and wire hang test. The foot-fault test was conducted according to the method described by Horiquini et al. [31]. The total number of footsteps and the number of fore- and hindlimb foot faults were recorded and used to calculate the foot fault rate. The cylinder test was conducted according to the method described by Tajiri et al. [32]. The number of forepaw contacts to the cylinder wall was recorded to calculate the contralateral paw use rate. The wire hang test was conducted similarly to the method described by Ahmed et al. [33] to measure grasping ability and forelimb strength. Each trial was evaluated on a 5-point scale. Two trials were performed for each rat on each testing day. At 14 days post-injury, the Morris water maze study was performed after the motor deficits of all rats had recovered. The water maze was filled with water (21–23 °C). The rats were first trained for 5 days with the platform located in the same position. The duration used to find the platform (escape latency) and the travel length before they arrived at the platform was recorded once per day for five consecutive days. The swimming speed was calculated from the swimming duration and travel length. 2.4. Nissl staining
2. Materials and methods At 7 days post-injury, rats were sacrificed and the brain was collected and fixed in 4% paraformaldehyde. On the next day, the fixed tissues were dehydrated and embedded in paraffin. They were then cut into serial 5-μm-thick sections. The cortex sections of 2.5–2.5 mm posterior to the bregma in the injured hemisphere were collected; the CA1 sections of hippocampus in the injured hemisphere were collected. On the day of Nissl staining, the sections of cortex and hippocampus were first immersed in an ethanol/chloroform solution and dehydrated in alcohol. Next, the sections were stained with 1% toluidine blue and differentiated in 95% alcohol. Afterwards, they were dehydrated in 100% alcohol and mounted with neutral gum. Nissl-positive cells were counted in five randomly selected horizons at 200 × magnification.
2.1. Animals Adult male Sprague-Dawley rats (body weight 150–180 g) were provided by the Animal Center of Daping Hospital of the Army Medical University and kept in clean stainless steel cages. They were allowed free access to food and water, and housed at room temperature under normal humidity (60 ± 10%). After the rats were adaptively fed for 7 days, they were divided into Control, Sham, TBI + Vehicle, TBI + MINO-10, TBI + MINO-20, TBI + MINO-40, TBI + Deferiprone and MINO-20 groups based on body weight. Rats in the Control group received no treatment. Rats in the Sham group received sham surgery 2
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mixture to obtain CAS indicator solution. Finally, the 0.5 ml CAS indicator solution was mixed with different concentrations of minocycline solution (0, 20, 40, 80, 160, 320, 640, 1280, 2560 μM). The absorbance was measured at 630 nm.
2.5. Stereological quantification The Stereological quantification was performed with the method described by Aronsson et al. [34]. Briefly, the sections of the injured hemisphere were stained with 0.5% cresyl violet (Sigma-Aldrich, USA). The neurons with a clearly visible nucleus and nucleolus were counted using an optical disector in five randomly chosen areas in three sections. The total number of neurons was estimated as N = ∑Q- × 1/ TSF × 1/ASF × 1/SSF (∑Q−: the total number of neurons counted within the dissector; TSF: the thickness sampling fraction, which means the ratio between the height of the disector and the section thickness; ASF: the area sampling fraction, which means the area of the counting frame relative to the area associated with movement to the next counting frame; SSF: the samples of a known fraction represent the section sampling fraction).
2.9. Western blotting After rats were sacrificed at 7 days post-injury, hippocampus and cortex tissue was collected for protein extraction. Proteins then underwent 12% SDS-PAGE and were transferred to PVDF membranes. The next day, the proteins were incubated with primary antibodies against ferritin, TfR1, DMT1, FPN1, and hepcidin at 37 °C for 2 h, and then with secondary antibodies for 2 h. The blots were examined using the BeyoECL Plus kit (Beyotime, Shanghai, China). 2.10. Statistical analysis
2.6. Measurement of iron concentrations in CSF and serum Data are presented as means ± SEM and were analyzed using oneway analysis of variance and the F-test, followed by the Student–Newman–Keuls post-hoc test. Statistical analysis was performed using SPSS 17.0 software. A P-value < 0.05 was considered to indicate statistical significance.
Iron concentrations in CSF and serum were measured at 7 days postinjury using a previously described method [35]. Briefly, after rats were sacrificed, CSF was collected from the cisterna magna by needle aspiration through the scalp. Blood was obtained from the main abdominal artery and allowed to clot at 25 °C for 1 h. Next, the blood was centrifuged at 1,600×g for 10 min at 25 °C to obtain the serum. A ferrozine reagent kit (Ferrozine, Elitech, Sées, France) was used to measure Fe3+ concentrations.
3. Results 3.1. Effects of TBI model on the brain tissue Fig. 1a shows the H&E staining results of the injured cortex of 3.0 mm posterior to the bregma in the injured and contralateral hemispheres. In the injured hemisphere, there is significant hemorrhage, which may be the source of the iron in the brain. In the contralateral hemisphere, no hemorrhage was observed, indicating that the hemorrhage is limited in the impact site.
2.7. Measurement of iron concentrations in brain tissues To measure the iron concentration, the cortex was collected from the area surrounding +3.0 mm posterior; 2.7 mm lateral to the bregma in the injured hemisphere. The whole hippocampus in the injured hemisphere was collected. Graphite furnace atomic absorption spectrometry was used to determine the iron content in the samples. Brain tissues were accurately weighed and added into nitric acid/perchloric acid solution for digestion for 10 h in the dark. The solution was then heated to boiling until it became transparent. After the remaining acid was volatilized by heating, the solution was cooled and deionized water was added. The solution was then heated again to dryness. Afterwards, 2% HNO3 was added to dissolve the residue. The Fe standard solution (1,000 pg/ml, National Standard Material Research Center, Beijing, China) was added to 2% HNO3 to obtain the desired concentrations (10 μg/L, 20 μg/L, 30 μg/L, and 40 μg/L). A standard curve was calculated by measuring the absorbance values of different concentrations of Fe standard solution. Finally, the absorbance value of the sample tube was measured to determine the iron concentrations in brain tissues.
3.2. Effects of minocycline on weight changes post-injury The weight changes of rats post-injury were recorded for 14 days and are shown in Fig. 1b and c. Fig. 1b shows the effects of different concentrations of minocycline on weight changes. 10 mg/kg minocycline had no significant effect on weight changes, but 20 mg/kg and 40 mg/kg minocycline significantly increased the body weight compared to TBI + Vehicle. Fig. 1c shows the effects of sham treatment, deferiprone, minocycline alone and 20 mg/kg minocycline on weight changes. Sham or minocycline treatment alone had no effect on weight change. The weights of rats in the TBI + Vehicle group significantly decreased after TBI surgery (P < 0.05 compared to Sham). When rats received TBI followed by daily minocycline or deferiprone treatment, their weights also dropped, but not as dramatically as in the TBI + Vehicle group. The difference between the TBI + Vehicle group and the TBI + MINO-20 or TBI + Deferiprone group was significant (P < 0.05).
2.8. Fe2+- and Fe3+- chelating activity measurement Briefly, Fe2+-chelating activity was measured by inhibition of the formation of iron (II)-ferrozine complex after pre-incubation of the reaction mixture with minocycline. The reaction mixture contained different concentrations of minocycline solution (0, 20, 40, 80, 160, 320, 640, 1280, 2560 μM), FeCl2 (0.6 mM) and methanol. It was well shaken and left at 25 °C for 10 min. Afterwards, 100 μl of ferrozine (5 mM in methanol) was added and mixed. The absorbance of the Fe2+–ferrozine complex was measured at 562 nm against methanol blanks. Fe3+-chelating activity measurement was performed with modified chrome azurol S (CAS) method. Briefly, 10 mmol/L hexadecyldimethylammonium bromide in distilled water, 1 mmol/L FeCl3 in 10 mmol/L HCI and 2 mmol/L CAS in distilled water was prepared. Next, 4.3 g of piperazine was dissolved in 20 ml distilled water. 6 ml of hexadecyldimethylammonium bromide and 1.5 ml of FeCl3 were added into 20 ml distilled water and mixed well, then 7.5 ml CAS was added and mixed again. Next, the piperazine solution was added to the
3.3. Effects of minocycline on neurological impairment The effects of minocycline on neurological impairment were assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Fig. 2 shows the results of the foot-fault test (2a and b), cylinder test (2c and d). 10 mg/kg minocycline had no significant effect on foot fault rate or contralateral paw use rate, but 20 mg/kg and 40 mg/kg minocycline significantly decreased the foot fault rate and increased the contralateral paw use rate compared to TBI + Vehicle. The foot fault rate in the TBI + MINO-20 or TBI + Deferiprone group was significantly lower than that in the TBI + Vehicle group (P < 0.05), whereas the contralateral paw use rate in the TBI + MINO-20 or TBI + Deferiprone group was significantly higher than that in the TBI + Vehicle group (P < 0.05). The scores in the wire hang tests of 3
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Fig. 1. Effects of minocycline on weight change after TBI. Body weights of rats were measured daily beginning at 1 day post-injury and continuing for 14 days. Weight changes were calculated from the body weights of rats on the day of TBI. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO-10: TBI surgery and 10 mg/kg minocycline injection; TBI + MINO-20: TBI surgery and 20 mg/kg minocycline injection; TBI + MINO-40: TBI surgery and 40 mg/kg minocycline injection; MINO-20: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
Fig. 2. Effects of minocycline on foot-fault test and cylinder test. The foot-fault test (a–b) and cylinder test (c–d) were performed daily beginning at 7 day post-injury and continuing for 8 days. The scores of wire hang test were 5.0 in all the groups after 7 day post-injury (data not shown). Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO-10: TBI surgery and 10 mg/kg minocycline injection; TBI + MINO-20: TBI surgery and 20 mg/kg minocycline injection; TBI + MINO-40: TBI surgery and 40 mg/kg minocycline injection; MINO-20: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group. 4
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Fig. 3. Effects of minocycline on Morris water maze outcomes. At 14 days post-injury, the Morris water maze study was performed. Changes in travel length (a–b), escape latency (c–d) and swimming speed (e–f) on each day. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO-10: TBI surgery and 10 mg/ kg minocycline injection; TBI + MINO-20: TBI surgery and 20 mg/kg minocycline injection; TBI + MINO-40: TBI surgery and 40 mg/kg minocycline injection; MINO-20: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; # P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
of Nissl-positive cells in the hippocampus and cortex of the TBI + Vehicle group were significantly lower than in the Sham group (P < 0.05), whereas minocycline treatment significantly attenuated the TBI-induced decrease in Nissl-positive cells (P < 0.05).
rats in all groups were 5.0 (data not shown). Sham or minocycline treatment alone had no impact on the results of the foot-fault test, cylinder test, or wire hang test. Fig. 3 shows the results of the Morris water maze study. Fig. 3a and b shows the changes in travel length on each day. On day 4 and 5 postinjury, 10 mg/kg minocycline had no significant effect on travel length, but 20 mg/kg and 40 mg/kg minocycline significantly decreased the travel length compared to TBI + Vehicle. On day 4 and 5 post-injury, the travel lengths of rats in the TBI + MINO-20 or TBI + Deferiprone group were significantly shorter than those of rats in the TBI + Vehicle group (P < 0.05). As shown in Fig. 3c and d, on days 3, 4, and 5 post-injury, 10 mg/kg minocycline had no significant effect on escape latency, but 20 mg/kg and 40 mg/kg minocycline significantly decreased the escape latency compared to TBI + Vehicle. The escape latency of rats in the TBI + MINO-20 or TBI + Deferiprone group was significantly lower than that of rats in the TBI + Vehicle group (P < 0.05). Fig. 3e and f shows the swimming speeds of rats each day. There was no significant difference between groups in the mean speed, indicating that motor deficit was not present in rats. Sham or minocycline treatment alone had no effects on neurological outcomes. Sham or minocycline treatment alone had no impact on the results of Morris water maze study.
3.5. Iron concentrations in CSF, serum, and brain tissues and the Fe2+- and Fe3+- chelating activity of minocycline Fig. 5 shows the iron concentrations in CSF and serum. As shown in Fig. 5a, the iron concentration in CSF was significantly increased in the TBI + Vehicle group (P < 0.05), whereas minocycline treatment significantly attenuated this increase (P < 0.05 compared to TBI + Vehicle). As shown in Fig. 5b, the iron concentration in serum did not significantly differ among the five groups. The iron concentrations in brain tissues (cortex, hippocampus, and whole brain) are shown in Table 1. Iron concentrations were significantly enhanced by TBI in the cortex (P < 0.05 compared to Sham) and hippocampus (P < 0.05 compared to Sham), but this increase was significantly attenuated by minocycline (P < 0.05 compared to TBI + Vehicle). Sham or minocycline treatment alone had no effect on iron concentration. The iron concentrations in whole brain tissue did not significantly differ among the five groups. The Fe2+- and Fe3+- chelating activity of minocycline was shown in Fig. 5c–d. It was shown that as the concentration of minocycline increased, the absorbance value of Fe3+ and Fe2+ solution significantly decreased, which indicates that minocycline had Fe3+and Fe2+- chelating activity.
3.4. Effects of minocycline on cell viability in the brain To measure the effects of minocycline on cell viability in the brain, we measured the cell viability using stereological analysis and Nissl staining. The stereological analysis (Fig. 4a and b) showed that the total number of viable neurons in the hippocampus and cortex were significantly decreased in the TBI model rats (P < 0.05), which was partially recovered by minocyclein treatment (P < 0.05). The results of Nissl staining are shown in Fig. 4c–e. Fig. 4c shows representative images; Fig. 4d and e shows the ratios of Nissl-positive cells in the hippocampus and cortex, respectively. The results show that the ratios
3.6. Effects of minocycline on the expression of iron metabolism proteins To measure the effects of minocycline on the expression of iron metabolism proteins (ferritin, TfR1, DMT1, FPN1, and hepcidin) in the hippocampus and cortex, we analyzed their protein levels by western blotting. As shown in Fig. 6a and b, the protein levels of ferritin and TfR1 in the TBI + Vehicle group were significantly higher than those in the Sham group (P < 0.05). Their expression levels in the TBI + MINO 5
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Fig. 4. Effects of minocycline on cell viability assessed by stereological analysis and Nissl staining. (a) Cell counts in the hippocampus; (b) Cell counts in the cortex; (c) Representative images of Nissl staining in hippocampus and cortex; (d) Rates of Nissl-positive cells in hippocampus; (e) Rates of Nissl-positive cells in cortex. Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and 20 mg/kg minocycline injection; MINO: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
of iron metabolism in the neuroprotective action of minocycline. First of all, the H&E staining results of the injured hemispheres showed significant hemorrhage, indicating the success of TBI model. The hemorrhage may be the source of the iron in the brain. In the contralateral hemisphere, no hemorrhage was observed, indicating that the hemorrhage is limited in the impact site. Next, our results showed that minocycline attenuated the neurological impairment caused by TBI, as demonstrated by foot-fault test, cylinder test, and Morris water maze. It is interesting to note that at 7 days post TBI injury, the scores of hang wire tests were 5.0 in all the groups. As the hang wire tests assess grasping ability and forelimb strength, foot-fault tests assess the locomotor function, and cylinder tests assess the locomotor asymmetry,
group, however, were significantly decreased (P < 0.05). The expression of DMT1 did not significantly differ among the groups. The protein levels of FPN1 were significantly decreased in the TBI + Vehicle group, but recovered in the TBI + MINO group. By contrast, the protein levels of hepcidin were significantly increased in the TBI + Vehicle group, and recovered in the TBI + MINO group. Sham or minocycline treatment alone had no effect on the expression of these proteins.
4. Discussion In the present study, we used a rat model of TBI to explore the role 6
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Fig. 5. Effects of minocycline on iron concentrations in CSF and serum and the Fe2+- and Fe3+chelating activity of minocycline. (a) Iron concentration in CSF. (b) Iron concentrations in CSF and serum. (c–d) Fe2+- and Fe3+- chelating activity of minocycline. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and 20 mg/kg minocycline injection; MINO: 20 mg/kg minocycline injection only. CSF: cerebrospinal fluid; TBI: traumatic brain injury; MINO: minocycline; # P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
while increasing neurodegeneration in TBI model. The microglial reactivity and neurodegeneration were found to be exacerbated by minocycline in TBI of neonate rat, suggesting minocycline may not be effective for TBI in the immature brain [42]. The present study focused on the effect of minocycline on neurological impairment caused by TBI. Consistent with previous studies, we found that minocycline significantly attenuated the decrease in body weight. The foot fault rate was significantly decreased by minocycline, whereas the contralateral paw use rate were significantly improved by minocycline treatment. In the Morris water maze study, the travel lengths and escape latency in the TBI + MINO-20 group were significantly decreased compared to those of rats in the TBI + Vehicle group. These parameters suggest a protective effect of minocycline against neurological impairment caused by TBI. Furthermore, Stereological analysis and Nissl staining in rat hippocampus and cortex showed that the cell viability was significantly increased in both tissues by minocycline, indicating the protective effect of minocycline on cell survival. Oxidative damage may play a key role in TBI. After TBI, a large number of red blood cells in the wound can be dissolved, which releases a large amount of heme and iron ions, causing neurotoxicity, brain atrophy, and neurological impairment [43]. We found that the iron concentrations in CSF and brain tissues (cortex and hippocampus) were significantly decreased by minocycline, suggesting that minocycline could reduce the iron concentration in the brain. It is interesting to note that the iron concentrations in the cortex and hippocampus of the injured area were higher than the iron concentration of the whole brain. Previous studies have suggested that after TBI, a large number of red
these results indicate that the grasping ability and forelimb strength of rats recovered more quickly than the locomotor function and locomotor asymmetry. Stereological analysis and Nissl staining results showed that cell viability in the brain tissue was greatly enhanced by minocycline. Further investigation revealed the effect of minocycline in reducing iron concentrations in CSF and brain tissues (cortex and hippocampus). Finally, we found that minocycline regulated the expression of iron metabolism proteins (ferritin, FPN1, and hepcidin). Studies have shown that minocycline is protective against brain damage [36,37]. Much evidence has suggested that minocycline has neuroprotective activity in traumatic brain injury. Meythaler et al. [38] tested the safety and feasibility of minocycline in treatment of acute traumatic brain injury patients and found that minocycline was safe for moderate to severe TBI at a dose twice that as recommended for treatment of infection. A Phase II minocycline trial was conducted to examine its effect against human spinal cord injury and found that HO1 and neurofilament heavy chain in the cerebrospinal fluid were changed by minocycline treatment [39]. Sangobowale et al. [40] treated mice with closed head injury with minocycline plus N-acetylcysteine (NAC) 12 h later and revealed that MINO plus NAC or MINO alone when first dosed 12 h after CHI increased myelin content using similar mechanisms seen when first dosed 1 h after closed head injury, suggesting that drugs protect oligodendrocytes with a clinically useful therapeutic time window. Minocycline could also attenuate TBI injury in post-natal Day 17 rats [41]. However, there are also some confusing results regarding the protective effect of MINO against TBI. Scott et al. [26] found out that minocycline reduced chronic microglial activation Table 1 Iron concentration in the brain of rats (μg/g tissue).
Cortex Hippocampus Whole brain
Control
Sham
TBI + Vehicle
TBI + MINO
MINO
18.66 ± 3.18 40.28 ± 2.41 14.96 ± 2.58
17.89 ± 2.59 42.56 ± 3.69 15.45 ± 2.86
35.87 ± 3.17# 69.45 ± 4.99# 14.55 ± 3.59
25.45 ± 2.94* 51.85 ± 3.46* 13.54 ± 3.22
21.45 ± 2.31 42.87 ± 3.28 14.79 ± 3.39
The iron concentration in the cortex, hippocampus and whole brain of rats was measured. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and minocycline injection; MINO: minocycline injection only. TBI: traumatic brain injury; MINO: minocycline. #: p < 0.05 compared to Sham. *: p < 0.05 compared to TBI + Vehicle. N = 10 per group. 7
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Fig. 6. Effects of minocycline on the expression of iron metabolism proteins in the hippocampus and cortex. Protein levels of ferritin (a), TfR1 (b), DMT1 (b), FPN1 (c), and hepcidin (d). Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and 20 mg/kg minocycline injection; MINO: 20 mg/kg minocycline injection only. TfR1: transferrin receptor 1; DMT1: divalent metal transporter 1; FPN1: ferroportin 1; TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 6 per group.
in these organs. When iron in the blood increases, transferrin receptor (TfR)-mediated iron absorption in liver also increases, which leads to increased hepcidin synthesis and secretion. As the hepcidin concentration in the blood increases, more hepcidin arrives at the target organ and then suppresses the iron absorption in the small intestine. Meanwhile, it increases the iron uptake by liver cells, bone marrow cells and macrophages, thereby increasing iron storage and utilization. Through this hepcidin-mediated pathway, blood iron concentrations will return to normal levels. Recent studies have confirmed that the brain itself is capable of expressing hepcidin and hepcidin is widely distributed in the brain [46–48]. It was found that hepcidin was expressed in endothelium and pericytes of blood vessels, glial cells, olfactory bulb, sub-ventricular zone and dentate gyrus. It was also shown that hepcidin co-localised with FPN in ependymal cells of the subventricular zone and in the corpus callosum. A significant increase in the expression of iron uptake (TfR1 and DMT1) and release (Fpn1) proteins was found in astrocytes [49]. Previous studies have found Fpn1 in neurons, astrocytes, and oligodendrocytes, which indicated that Fpn1 might be essential in iron efflux. Tf in the brain can be produced in oligodendrocytes or epithelial cells of the choroid plexus and Tf-Fe can be taken up by neurons and astrocytes via a TfR1-mediated process [50]. Our results showed that FPN1 was significantly decreased in the TBI + Vehicle group, but recovered in the TBI + MINO group. By contrast, the protein levels of hepcidin were significantly increased in the TBI + Vehicle group, but decreased in the TBI + MINO group. This is probably a result of the increase in the iron concentration, which leads to increased hepcidin synthesis and secretion. These results indicate that minocycline may restore the expression of FPN1 by inhibiting the overexpression of hepcidin. Furthermore, it is interesting to note that minocycline treatment reduced brain iron accumulation and suppressed iron metabolizing protein upregulation, but did not alter the normal level of iron or iron metabolizing proteins in healthy rats. Our study has showed that minocycline has strong iron-chelating activity. It is possible that the difference between the state of iron of TBI rats and healthy rats may lead to the iron-chelating activity of minocycline; therefore minocycline treatment reduced brain iron accumulation in TBI rats, but did not alter the normal level of iron in sham treated rats. Regarding the iron metabolizing proteins, it is possible that the change of iron metabolizing proteins is a response to the change of iron level in the brain. Minocycline may don't directly change the expression of
blood cells in the wound can be dissolved, which releases a large amount of heme and iron ions, causing neurotoxicity, brain atrophy, and neurological impairment [43], which may be the reason that the local iron concentration were increased, but the overall iron concentration did not increase to a significant level. The iron concentrations in serum, however, were not affected by minocycline, indicating that the iron-reducing effect of minocycline was limited locally. The in vitro study showed that minocycline could directly bind Fe2+ or Fe3+, indicating that directly reducing the iron concentration may be a mechanism of minocycline. In the present study, a significant increase in ferritin expression was observed in the TBI + Vehicle group, suggesting that ferritin was upregulated in the brain tissue to increase iron ion storage to avoid damage from iron ions. Treatment with minocycline significantly decreased ferritin levels in the brain tissue, probably because it significantly reduced the level of iron. The fact that minocycline treatment alone did not change the ferritin level suggests that minocycline did not directly regulate ferritin expression. The Tf/TfR pathway is the main route of iron transport. Xi et al. [13] found that ferritin was upregulated after experimental intracerebral hemorrhage, and the uptake of iron by cells increased, resulting in neurotoxicity. The level of TfR1 of the TBI + Vehicle group in the hippocampus and cortex was significantly higher than that in the Sham group. Its expression in the TBI + MINO group, however, was significantly decreased compared to that in the TBI + Vehicle group. This result suggests that ferritin is also upregulated after TBI, causing neurotoxicity, but treatment with minocycline can inhibit its increase, thus inhibiting TBI-induced neurotoxicity. However, the expression of another important iron metabolism protein, DMT1, was not affected by the TBI model or minocycline, indicating that DMT1 may not be involved. To further investigate the mechanism by which minocycline regulates iron metabolism, we examined the expression levels of FPN1 and hepcidin. Studies have shown that FPN1 is essential for iron export from neurons [44]. Hepcidin is a major regulator of the activity of FPN1, which is mainly expressed in astrocytes [45]. Increased levels of hepcidin can lead to internalization and degradation of FPN1 by binding to its evolutionarily conserved hepcidin-binding domain, which eventually causes iron accumulation in neurons [44]. Hepcidin is mainly synthesized in the liver and then secreted into the blood. It is transported to its main target organs, including the small intestine, liver, bone marrow cells and macrophages, and controls the iron metabolism 8
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these iron metabolizing proteins, but can alter their expression by chelating excess iron in the TBI. As a result, it inhibited the over expression of iron metabolizing proteins in the TBI rats, but did not change the normal level of iron metabolizing proteins in the sham treated rats. In conclusion, the present study shows that minocycline significantly attenuated the neurological impairment caused by TBI, and increased neuronal viability. Minocycline reduced the iron concentrations in CSF and brain tissues (cortex and hippocampus) and had ironchelating activity. Minocycline also inhibited the overexpression of ferritin and TfR1 in the hippocampus and cortex, but did not affect the expression of DMT1. Minocycline restored the expression of FPN1 by decreasing the expression of hepcidin. Taken together, these results suggest that minocycline attenuates neurological impairment caused by TBI and regulates iron metabolism.
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CRediT authorship contribution statement Lijun Zhang: Methodology, Validation, Investigation, Funding acquisition, Writing - original draft. Hong Xiao: Formal analysis, Writing - review & editing. Xing Yu: Resources, Data curation. Yongbing Deng: Conceptualization, Funding acquisition. Declaration of competing interest There is no conflict of interest. Acknowledgements This work was supported by Research on Basic Science and Advanced Techniques in Chongqing (cstc2015jcyjA10098), Science and Technology Project of Taizhou, Zhejiang, China (1901ky57) and Research on Basic Science and Advanced Techniques of Yuzhong District, Chongqing (20170139). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.abb.2020.108302. References [1] M.A. Vella, M.L. Crandall, M.B. Patel, Acute management of traumatic brain injury, Surg. Clin. North Am. 97 (2017) 1015–1030. [2] M. Galgano, G. Toshkezi, X. Qiu, T. Russell, L. Chin, L.R. Zhao, Traumatic brain injury: current treatment strategies and future endeavors, Cell Transplant. 26 (2017) 1118–1130. [3] A.I. Maas, N. Stocchetti, R. Bullock, Moderate and severe traumatic brain injury in adults, Lancet Neurol. 7 (2008) 728–741. [4] X. Wu, J. Hu, L. Zhuo, C. Fu, G. Hui, Y. Wang, W. Yang, L. Teng, S. Lu, G. Xu, Epidemiology of traumatic brain injury in eastern China, 2004: a prospective large case study, J. Trauma 64 (2008) 1313–1319. [5] D.K. Menon, Unique challenges in clinical trials in traumatic brain injury, Crit. Care Med. 37 (2009) S129–S135. [6] S. Rooker, S. Jander, J. Van Reempts, G. Stoll, P.G. Jorens, M. Borgers, J. Verlooy, Spatiotemporal pattern of neuroinflammation after impact-acceleration closed head injury in the rat, Mediat. Inflamm. 1 (2006) 90123. [7] H.J. Thompson, N. Marklund, D.G. LeBold, D.M. Morales, C.A. Keck, M. Vinson, N.C. Royo, R. Grundy, T.K. McIntosh, Tissue sparing and functional recovery following experimental traumatic brain injury is provided by treatment with an antimyelin-associated glycoprotein antibody, Eur. J. Neurosci. 24 (2006) 3063–3072. [8] S. Altamura, M.U. Muckenthaler, Iron toxicity in diseases of aging: Alzheimer's disease, Parkinson's disease and atherosclerosis, J. Alzheim. Dis. 16 (2009) 879–895. [9] Y. Ke, Z.M. Qian, Brain iron metabolism: neurobiology and neurochemistry, Prog. Neurobiol. 83 (2007) 149–173. [10] D.A. Long, K. Ghosh, A.N. Moore, C.E. Dixon, P.K. Dash, Deferoxamine improves spatial memory performance following experimental brain injury in rats, Brain Res. 717 (1996) 109–117. [11] J. Zhao, Z. Chen, G. Xi, R.F. Keep, Y. Hua, Deferoxamine attenuates acute hydrocephalus after traumatic brain injury in rats, Transl. Stroke Res. 5 (2014) 586–594. [12] Y. Hua, T. Nakamura, R.F. Keep, J. Wu, T. Schallert, J.T. Hoff, G. Xi, Long-term effects of experimental intracerebral hemorrhage: the role of iron, J. Neurosurg.
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Minocycline attenuates neurological impairment and regulates iron metabolism in a rat model of traumatic brain injury
T
Lijun Zhanga, Hong Xiaob, Xing Yub, Yongbing Dengb,∗ a b
Department of Neurosurgery, First People's Hospital of Taizhou, Taizhou, Zhejiang, 318020, China Department of Neurosurgery, The Fourth People's Hospital of Chongqing, 400014, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Traumatic brain injury Minocycline Neurological impairment Iron metabolism
There is currently no effective treatment for neurological impairment caused by traumatic brain injury (TBI). It has been reported that excessive iron production in the brain may be a key factor in neurological impairment. In the present study, we investigated the effects of minocycline, a semi-synthetic tetracycline antibiotic, against TBI-induced neurological impairment and explored its underlying mechanism. Neurological impairment was assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Nissl staining was performed to evaluate cell viability in the brain. The iron concentrations in cerebrospinal fluid (CSF), serum, and brain tissues were examined. The Fe2+- and Fe3+- chelating activity of minocycline was measured. Finally, the expression levels of important iron metabolism proteins ferritin, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hepcidin in the hippocampus and cortex were measured by Western blot analysis. The results indicate that minocycline significantly attenuated the neurological impairment caused by TBI and increased neuronal viability. Minocycline showed a Fe2+- and Fe3+- chelating activity in vitro and reduced the iron concentration in CSF and brain tissues (cortex and hippocampus). Minocycline also inhibited the overexpression of ferritin and TfR1, but did not affect the expression of DMT1. Minocycline restored the expression of FPN1 by decreasing the expression of hepcidin. In conclusion, minocycline may attenuate neurological impairment caused by TBI and regulate iron metabolism.
1. Introduction Traumatic brain injury (TBI) is a major cause of trauma, which is responsible for the majority of death in individuals aged 1–45 [1]. According to a survey conducted in 2016, the rates of emergency department visits, hospitalizations, and deaths related to TBI have significantly increased from 2001 to 2010 [2]. The incidence of TBI has rapidly increased due to traffic accidents, wars, accidents, and natural disasters [3]. Approximately 60% of TBI cases and 70% of severe head trauma cases are caused by car accidents in China [4]. With the development of modern medical technology, the mortality rate of TBI patients has significantly decreased, but many patients have long-term motor, sensory, behavioral, and cognitive dysfunction [3]. For secondary injuries caused by TBI, such as axonal rupture, neuronal apoptosis, brain atrophy, and glial scar formation, and subsequent behavioral and cognitive dysfunction, no effective treatment is yet available. At present, the pathological mechanisms involved in secondary injury of TBI include neuronal apoptosis, inflammation, oxidative
∗
damage, and disorders of energy metabolism [5–7]. Many studies have shown that overproduction of iron in the brain has a neurotoxic effect and is involved in neurodegenerative diseases and intracerebral hemorrhage [8,9]. Studies have reported that intracerebroventricular injection of autologous blood and FeCl3 induced chronic hydrocephalus, whereas treatment with an iron-chelating agent (deferoxamine; DFO) reduced the incidence and severity of hydrocephalus and improved Morris water maze test results [10,11]. These results suggest that excessive iron in the ventricle has a neurotoxic effect, and DFO can attenuate the neurotoxicity by reducing excessive iron. Studies of intracerebral hemorrhage [12,13] and neurodegenerative disease [14] revealed that excessive iron may be essential in brain atrophy and neurological impairment. An increase in brain iron content was observed 1 day after experimental intracerebral hemorrhage and lasted for 3 months [12]. Xi et al. found that injecting Fe3+ into brain parenchyma can cause brain parenchymal injury [13]. Minocycline, a semi-synthetic tetracycline antibiotic, has good lipid solubility and can easily pass through the blood–brain barrier. Experimental studies have found that minocycline can protect neurons
Corresponding author. Department of Neurosurgery, The Fourth People's Hospital of Chongqing, Chongqing, 400014, China. E-mail address: [email protected] (Y. Deng).
https://doi.org/10.1016/j.abb.2020.108302 Received 18 September 2019; Received in revised form 8 February 2020; Accepted 8 February 2020 Available online 10 February 2020 0003-9861/ © 2020 Published by Elsevier Inc.
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similar to TBI. Rats in the TBI + Vehicle group received TBI treatment and intraperitoneal (i.p.) saline injection. Rats in the TBI + MINO-10, TBI + MINO-20, TBI + MINO-40 group received TBI treatment and i.p. injection of 10 mg/kg, 20 mg/kg, and 40 mg/kg of minocycline. Rats in the TBI + Deferiprone group received TBI treatment and i.p. injection of deferiprone (50 mg/kg, Apopharma, Inc., Canada). Rats in the MINO20 group received i.p. injection of 20 mg/kg minocycline only. Daily injection of minocycline (Sigma, St. Louis, MO, USA) started at 12 h post-injury and continued for 7 days. Rat body weights were measured daily post-injury. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition (National Academies Press, Washington, DC, 2010).
against intracerebral hemorrhage, ischemia, brain trauma, and Alzheimer's disease [15–19]. It is generally believed that its effects include inhibition of microglia activation and inflammation through its anti-oxidant and anti-apoptotic actions [20–22]. Minocycline is also reported to inhibit metalloproteases, inducible nitric oxide synthase, and poly (ADP-ribose) polymerase [23,24], but the exact mechanism is not yet clear. On the other hand, although there are many studies that have found beneficial effects of minocycline treatment following TBI, there are also mixed results, with some studies showing minimal or no beneficial effects. For example, Sanuki et al. found that minocycline did not increase the survival of aged penetrating TBI flies [25]. Scott revealed that minocycline reduced chronic microglia activation but increased neurodegeneration in a TBI model [26]. Therefore, it remains significative to examine the effect of minocycline on different markers of TBI. In brain tissue, most iron ions bind to proteins in the form of stable ferritin. Ferritin consists of two different homologous proteins, ferritin light chain (FL) and ferritin heavy chain (FH). Transferrin (Tf) is a single chain polypeptide with a molecular weight of 75 kg/mol. First, iron ions in the cerebrospinal fluid (CSF)/brain interstitial fluid bind to Tf and form a complex with transferrin receptor (TfR). The complex forms a phagocytic vacuole and enters the cell, in which Fe3+ reductase deoxidizes Fe3+ to Fe2+, which is then transported to the cytoplasm and participates in cellular functions. After the release of iron ions, the Tf/TfR complex returns to the cell surface, where Tf is released to the extracellular matrix. TfR is mainly located in oligodendrocytes, suggesting that these cells take in ferric ion mainly through the Tf/TfR pathway. The main function of divalent metal transporter 1 (DMT1) is to mediate the entry of Fe2+ into the cell and transport Fe2+ from the endocytic vesicles into the cytoplasm. A G185R mutation in the DMT1 gene may cause severe iron deficiency anemia in mice [27]. DMT1 is closely related to human diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease [28]. Ferroportin 1 (FPN1), also known as iron-regulated transporter 1 (IREG1) or metal transporter protein 1 (MTP1), is a transmembrane iron export protein. The main function of the FPN1 protein is to transport iron and manganese in the cell into the plasma, releasing iron from the cell [29]. FPN1 is highly expressed in rat hippocampus, cerebral cortex, cerebellum, thalamus, and striatum. Hepcidin acts as an important protein regulating the expression of FPN1 [29]. Whether and how minocycline regulates these proteins attracted our curiosity. In the present study, we used a rat model of TBI, a weight drop model, to explore the role of iron metabolism in the neuroprotective action of minocycline. The effect of minocycline on neurological impairment was assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Nissl staining was performed to evaluate cell viability in the brain tissue. The Fe2+- and Fe3+- chelating activity of minocycline was measured. The iron concentrations in CSF, serum, and brain tissues were examined. Finally, several important iron-related proteins, ferritin, TfR1, DMT1, FPN1, and hepcidin, were measured by Western blot analysis to explore the effect of minocycline on iron metabolism.
2.2. Rat model of focal TBI The TBI procedure, a weight drop model, was similar to that described by Li et al. [30]. First, rats were given ketamine (90 mg/kg, ip) and xylazine (10 mg/kg, ip) for complete anesthesia. Next, the skull was exposed after a midline scalp incision. A left parietal bone window was made using a dental drill (+3.0 mm posterior; 2.7 mm lateral to the bregma). Trauma was inflicted by a cylindrical steel rod (diameter, 4 mm; weight, 40 g) that fell from a height of 25 cm onto a piston on the surface of the dura mater. Finally, the incision was closed and lidocaine jelly was used to prevent infection. Rats in the Sham group were subjected to the same procedure, except the cylindrical steel rod did not fall. H&E staining of the cortex of 3.0 mm posterior to the bregma in the injured and contralateral hemispheres was performed to show the impact site. 2.3. Assessment of neurological impairment At 7 days post-injury, neurological impairment was assessed once daily by foot-fault test, cylinder test, and wire hang test. The foot-fault test was conducted according to the method described by Horiquini et al. [31]. The total number of footsteps and the number of fore- and hindlimb foot faults were recorded and used to calculate the foot fault rate. The cylinder test was conducted according to the method described by Tajiri et al. [32]. The number of forepaw contacts to the cylinder wall was recorded to calculate the contralateral paw use rate. The wire hang test was conducted similarly to the method described by Ahmed et al. [33] to measure grasping ability and forelimb strength. Each trial was evaluated on a 5-point scale. Two trials were performed for each rat on each testing day. At 14 days post-injury, the Morris water maze study was performed after the motor deficits of all rats had recovered. The water maze was filled with water (21–23 °C). The rats were first trained for 5 days with the platform located in the same position. The duration used to find the platform (escape latency) and the travel length before they arrived at the platform was recorded once per day for five consecutive days. The swimming speed was calculated from the swimming duration and travel length. 2.4. Nissl staining
2. Materials and methods At 7 days post-injury, rats were sacrificed and the brain was collected and fixed in 4% paraformaldehyde. On the next day, the fixed tissues were dehydrated and embedded in paraffin. They were then cut into serial 5-μm-thick sections. The cortex sections of 2.5–2.5 mm posterior to the bregma in the injured hemisphere were collected; the CA1 sections of hippocampus in the injured hemisphere were collected. On the day of Nissl staining, the sections of cortex and hippocampus were first immersed in an ethanol/chloroform solution and dehydrated in alcohol. Next, the sections were stained with 1% toluidine blue and differentiated in 95% alcohol. Afterwards, they were dehydrated in 100% alcohol and mounted with neutral gum. Nissl-positive cells were counted in five randomly selected horizons at 200 × magnification.
2.1. Animals Adult male Sprague-Dawley rats (body weight 150–180 g) were provided by the Animal Center of Daping Hospital of the Army Medical University and kept in clean stainless steel cages. They were allowed free access to food and water, and housed at room temperature under normal humidity (60 ± 10%). After the rats were adaptively fed for 7 days, they were divided into Control, Sham, TBI + Vehicle, TBI + MINO-10, TBI + MINO-20, TBI + MINO-40, TBI + Deferiprone and MINO-20 groups based on body weight. Rats in the Control group received no treatment. Rats in the Sham group received sham surgery 2
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mixture to obtain CAS indicator solution. Finally, the 0.5 ml CAS indicator solution was mixed with different concentrations of minocycline solution (0, 20, 40, 80, 160, 320, 640, 1280, 2560 μM). The absorbance was measured at 630 nm.
2.5. Stereological quantification The Stereological quantification was performed with the method described by Aronsson et al. [34]. Briefly, the sections of the injured hemisphere were stained with 0.5% cresyl violet (Sigma-Aldrich, USA). The neurons with a clearly visible nucleus and nucleolus were counted using an optical disector in five randomly chosen areas in three sections. The total number of neurons was estimated as N = ∑Q- × 1/ TSF × 1/ASF × 1/SSF (∑Q−: the total number of neurons counted within the dissector; TSF: the thickness sampling fraction, which means the ratio between the height of the disector and the section thickness; ASF: the area sampling fraction, which means the area of the counting frame relative to the area associated with movement to the next counting frame; SSF: the samples of a known fraction represent the section sampling fraction).
2.9. Western blotting After rats were sacrificed at 7 days post-injury, hippocampus and cortex tissue was collected for protein extraction. Proteins then underwent 12% SDS-PAGE and were transferred to PVDF membranes. The next day, the proteins were incubated with primary antibodies against ferritin, TfR1, DMT1, FPN1, and hepcidin at 37 °C for 2 h, and then with secondary antibodies for 2 h. The blots were examined using the BeyoECL Plus kit (Beyotime, Shanghai, China). 2.10. Statistical analysis
2.6. Measurement of iron concentrations in CSF and serum Data are presented as means ± SEM and were analyzed using oneway analysis of variance and the F-test, followed by the Student–Newman–Keuls post-hoc test. Statistical analysis was performed using SPSS 17.0 software. A P-value < 0.05 was considered to indicate statistical significance.
Iron concentrations in CSF and serum were measured at 7 days postinjury using a previously described method [35]. Briefly, after rats were sacrificed, CSF was collected from the cisterna magna by needle aspiration through the scalp. Blood was obtained from the main abdominal artery and allowed to clot at 25 °C for 1 h. Next, the blood was centrifuged at 1,600×g for 10 min at 25 °C to obtain the serum. A ferrozine reagent kit (Ferrozine, Elitech, Sées, France) was used to measure Fe3+ concentrations.
3. Results 3.1. Effects of TBI model on the brain tissue Fig. 1a shows the H&E staining results of the injured cortex of 3.0 mm posterior to the bregma in the injured and contralateral hemispheres. In the injured hemisphere, there is significant hemorrhage, which may be the source of the iron in the brain. In the contralateral hemisphere, no hemorrhage was observed, indicating that the hemorrhage is limited in the impact site.
2.7. Measurement of iron concentrations in brain tissues To measure the iron concentration, the cortex was collected from the area surrounding +3.0 mm posterior; 2.7 mm lateral to the bregma in the injured hemisphere. The whole hippocampus in the injured hemisphere was collected. Graphite furnace atomic absorption spectrometry was used to determine the iron content in the samples. Brain tissues were accurately weighed and added into nitric acid/perchloric acid solution for digestion for 10 h in the dark. The solution was then heated to boiling until it became transparent. After the remaining acid was volatilized by heating, the solution was cooled and deionized water was added. The solution was then heated again to dryness. Afterwards, 2% HNO3 was added to dissolve the residue. The Fe standard solution (1,000 pg/ml, National Standard Material Research Center, Beijing, China) was added to 2% HNO3 to obtain the desired concentrations (10 μg/L, 20 μg/L, 30 μg/L, and 40 μg/L). A standard curve was calculated by measuring the absorbance values of different concentrations of Fe standard solution. Finally, the absorbance value of the sample tube was measured to determine the iron concentrations in brain tissues.
3.2. Effects of minocycline on weight changes post-injury The weight changes of rats post-injury were recorded for 14 days and are shown in Fig. 1b and c. Fig. 1b shows the effects of different concentrations of minocycline on weight changes. 10 mg/kg minocycline had no significant effect on weight changes, but 20 mg/kg and 40 mg/kg minocycline significantly increased the body weight compared to TBI + Vehicle. Fig. 1c shows the effects of sham treatment, deferiprone, minocycline alone and 20 mg/kg minocycline on weight changes. Sham or minocycline treatment alone had no effect on weight change. The weights of rats in the TBI + Vehicle group significantly decreased after TBI surgery (P < 0.05 compared to Sham). When rats received TBI followed by daily minocycline or deferiprone treatment, their weights also dropped, but not as dramatically as in the TBI + Vehicle group. The difference between the TBI + Vehicle group and the TBI + MINO-20 or TBI + Deferiprone group was significant (P < 0.05).
2.8. Fe2+- and Fe3+- chelating activity measurement Briefly, Fe2+-chelating activity was measured by inhibition of the formation of iron (II)-ferrozine complex after pre-incubation of the reaction mixture with minocycline. The reaction mixture contained different concentrations of minocycline solution (0, 20, 40, 80, 160, 320, 640, 1280, 2560 μM), FeCl2 (0.6 mM) and methanol. It was well shaken and left at 25 °C for 10 min. Afterwards, 100 μl of ferrozine (5 mM in methanol) was added and mixed. The absorbance of the Fe2+–ferrozine complex was measured at 562 nm against methanol blanks. Fe3+-chelating activity measurement was performed with modified chrome azurol S (CAS) method. Briefly, 10 mmol/L hexadecyldimethylammonium bromide in distilled water, 1 mmol/L FeCl3 in 10 mmol/L HCI and 2 mmol/L CAS in distilled water was prepared. Next, 4.3 g of piperazine was dissolved in 20 ml distilled water. 6 ml of hexadecyldimethylammonium bromide and 1.5 ml of FeCl3 were added into 20 ml distilled water and mixed well, then 7.5 ml CAS was added and mixed again. Next, the piperazine solution was added to the
3.3. Effects of minocycline on neurological impairment The effects of minocycline on neurological impairment were assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Fig. 2 shows the results of the foot-fault test (2a and b), cylinder test (2c and d). 10 mg/kg minocycline had no significant effect on foot fault rate or contralateral paw use rate, but 20 mg/kg and 40 mg/kg minocycline significantly decreased the foot fault rate and increased the contralateral paw use rate compared to TBI + Vehicle. The foot fault rate in the TBI + MINO-20 or TBI + Deferiprone group was significantly lower than that in the TBI + Vehicle group (P < 0.05), whereas the contralateral paw use rate in the TBI + MINO-20 or TBI + Deferiprone group was significantly higher than that in the TBI + Vehicle group (P < 0.05). The scores in the wire hang tests of 3
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Fig. 1. Effects of minocycline on weight change after TBI. Body weights of rats were measured daily beginning at 1 day post-injury and continuing for 14 days. Weight changes were calculated from the body weights of rats on the day of TBI. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO-10: TBI surgery and 10 mg/kg minocycline injection; TBI + MINO-20: TBI surgery and 20 mg/kg minocycline injection; TBI + MINO-40: TBI surgery and 40 mg/kg minocycline injection; MINO-20: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
Fig. 2. Effects of minocycline on foot-fault test and cylinder test. The foot-fault test (a–b) and cylinder test (c–d) were performed daily beginning at 7 day post-injury and continuing for 8 days. The scores of wire hang test were 5.0 in all the groups after 7 day post-injury (data not shown). Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO-10: TBI surgery and 10 mg/kg minocycline injection; TBI + MINO-20: TBI surgery and 20 mg/kg minocycline injection; TBI + MINO-40: TBI surgery and 40 mg/kg minocycline injection; MINO-20: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group. 4
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Fig. 3. Effects of minocycline on Morris water maze outcomes. At 14 days post-injury, the Morris water maze study was performed. Changes in travel length (a–b), escape latency (c–d) and swimming speed (e–f) on each day. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO-10: TBI surgery and 10 mg/ kg minocycline injection; TBI + MINO-20: TBI surgery and 20 mg/kg minocycline injection; TBI + MINO-40: TBI surgery and 40 mg/kg minocycline injection; MINO-20: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; # P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
of Nissl-positive cells in the hippocampus and cortex of the TBI + Vehicle group were significantly lower than in the Sham group (P < 0.05), whereas minocycline treatment significantly attenuated the TBI-induced decrease in Nissl-positive cells (P < 0.05).
rats in all groups were 5.0 (data not shown). Sham or minocycline treatment alone had no impact on the results of the foot-fault test, cylinder test, or wire hang test. Fig. 3 shows the results of the Morris water maze study. Fig. 3a and b shows the changes in travel length on each day. On day 4 and 5 postinjury, 10 mg/kg minocycline had no significant effect on travel length, but 20 mg/kg and 40 mg/kg minocycline significantly decreased the travel length compared to TBI + Vehicle. On day 4 and 5 post-injury, the travel lengths of rats in the TBI + MINO-20 or TBI + Deferiprone group were significantly shorter than those of rats in the TBI + Vehicle group (P < 0.05). As shown in Fig. 3c and d, on days 3, 4, and 5 post-injury, 10 mg/kg minocycline had no significant effect on escape latency, but 20 mg/kg and 40 mg/kg minocycline significantly decreased the escape latency compared to TBI + Vehicle. The escape latency of rats in the TBI + MINO-20 or TBI + Deferiprone group was significantly lower than that of rats in the TBI + Vehicle group (P < 0.05). Fig. 3e and f shows the swimming speeds of rats each day. There was no significant difference between groups in the mean speed, indicating that motor deficit was not present in rats. Sham or minocycline treatment alone had no effects on neurological outcomes. Sham or minocycline treatment alone had no impact on the results of Morris water maze study.
3.5. Iron concentrations in CSF, serum, and brain tissues and the Fe2+- and Fe3+- chelating activity of minocycline Fig. 5 shows the iron concentrations in CSF and serum. As shown in Fig. 5a, the iron concentration in CSF was significantly increased in the TBI + Vehicle group (P < 0.05), whereas minocycline treatment significantly attenuated this increase (P < 0.05 compared to TBI + Vehicle). As shown in Fig. 5b, the iron concentration in serum did not significantly differ among the five groups. The iron concentrations in brain tissues (cortex, hippocampus, and whole brain) are shown in Table 1. Iron concentrations were significantly enhanced by TBI in the cortex (P < 0.05 compared to Sham) and hippocampus (P < 0.05 compared to Sham), but this increase was significantly attenuated by minocycline (P < 0.05 compared to TBI + Vehicle). Sham or minocycline treatment alone had no effect on iron concentration. The iron concentrations in whole brain tissue did not significantly differ among the five groups. The Fe2+- and Fe3+- chelating activity of minocycline was shown in Fig. 5c–d. It was shown that as the concentration of minocycline increased, the absorbance value of Fe3+ and Fe2+ solution significantly decreased, which indicates that minocycline had Fe3+and Fe2+- chelating activity.
3.4. Effects of minocycline on cell viability in the brain To measure the effects of minocycline on cell viability in the brain, we measured the cell viability using stereological analysis and Nissl staining. The stereological analysis (Fig. 4a and b) showed that the total number of viable neurons in the hippocampus and cortex were significantly decreased in the TBI model rats (P < 0.05), which was partially recovered by minocyclein treatment (P < 0.05). The results of Nissl staining are shown in Fig. 4c–e. Fig. 4c shows representative images; Fig. 4d and e shows the ratios of Nissl-positive cells in the hippocampus and cortex, respectively. The results show that the ratios
3.6. Effects of minocycline on the expression of iron metabolism proteins To measure the effects of minocycline on the expression of iron metabolism proteins (ferritin, TfR1, DMT1, FPN1, and hepcidin) in the hippocampus and cortex, we analyzed their protein levels by western blotting. As shown in Fig. 6a and b, the protein levels of ferritin and TfR1 in the TBI + Vehicle group were significantly higher than those in the Sham group (P < 0.05). Their expression levels in the TBI + MINO 5
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Fig. 4. Effects of minocycline on cell viability assessed by stereological analysis and Nissl staining. (a) Cell counts in the hippocampus; (b) Cell counts in the cortex; (c) Representative images of Nissl staining in hippocampus and cortex; (d) Rates of Nissl-positive cells in hippocampus; (e) Rates of Nissl-positive cells in cortex. Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and 20 mg/kg minocycline injection; MINO: 20 mg/kg minocycline injection only. TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
of iron metabolism in the neuroprotective action of minocycline. First of all, the H&E staining results of the injured hemispheres showed significant hemorrhage, indicating the success of TBI model. The hemorrhage may be the source of the iron in the brain. In the contralateral hemisphere, no hemorrhage was observed, indicating that the hemorrhage is limited in the impact site. Next, our results showed that minocycline attenuated the neurological impairment caused by TBI, as demonstrated by foot-fault test, cylinder test, and Morris water maze. It is interesting to note that at 7 days post TBI injury, the scores of hang wire tests were 5.0 in all the groups. As the hang wire tests assess grasping ability and forelimb strength, foot-fault tests assess the locomotor function, and cylinder tests assess the locomotor asymmetry,
group, however, were significantly decreased (P < 0.05). The expression of DMT1 did not significantly differ among the groups. The protein levels of FPN1 were significantly decreased in the TBI + Vehicle group, but recovered in the TBI + MINO group. By contrast, the protein levels of hepcidin were significantly increased in the TBI + Vehicle group, and recovered in the TBI + MINO group. Sham or minocycline treatment alone had no effect on the expression of these proteins.
4. Discussion In the present study, we used a rat model of TBI to explore the role 6
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Fig. 5. Effects of minocycline on iron concentrations in CSF and serum and the Fe2+- and Fe3+chelating activity of minocycline. (a) Iron concentration in CSF. (b) Iron concentrations in CSF and serum. (c–d) Fe2+- and Fe3+- chelating activity of minocycline. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and 20 mg/kg minocycline injection; MINO: 20 mg/kg minocycline injection only. CSF: cerebrospinal fluid; TBI: traumatic brain injury; MINO: minocycline; # P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 10 per group.
while increasing neurodegeneration in TBI model. The microglial reactivity and neurodegeneration were found to be exacerbated by minocycline in TBI of neonate rat, suggesting minocycline may not be effective for TBI in the immature brain [42]. The present study focused on the effect of minocycline on neurological impairment caused by TBI. Consistent with previous studies, we found that minocycline significantly attenuated the decrease in body weight. The foot fault rate was significantly decreased by minocycline, whereas the contralateral paw use rate were significantly improved by minocycline treatment. In the Morris water maze study, the travel lengths and escape latency in the TBI + MINO-20 group were significantly decreased compared to those of rats in the TBI + Vehicle group. These parameters suggest a protective effect of minocycline against neurological impairment caused by TBI. Furthermore, Stereological analysis and Nissl staining in rat hippocampus and cortex showed that the cell viability was significantly increased in both tissues by minocycline, indicating the protective effect of minocycline on cell survival. Oxidative damage may play a key role in TBI. After TBI, a large number of red blood cells in the wound can be dissolved, which releases a large amount of heme and iron ions, causing neurotoxicity, brain atrophy, and neurological impairment [43]. We found that the iron concentrations in CSF and brain tissues (cortex and hippocampus) were significantly decreased by minocycline, suggesting that minocycline could reduce the iron concentration in the brain. It is interesting to note that the iron concentrations in the cortex and hippocampus of the injured area were higher than the iron concentration of the whole brain. Previous studies have suggested that after TBI, a large number of red
these results indicate that the grasping ability and forelimb strength of rats recovered more quickly than the locomotor function and locomotor asymmetry. Stereological analysis and Nissl staining results showed that cell viability in the brain tissue was greatly enhanced by minocycline. Further investigation revealed the effect of minocycline in reducing iron concentrations in CSF and brain tissues (cortex and hippocampus). Finally, we found that minocycline regulated the expression of iron metabolism proteins (ferritin, FPN1, and hepcidin). Studies have shown that minocycline is protective against brain damage [36,37]. Much evidence has suggested that minocycline has neuroprotective activity in traumatic brain injury. Meythaler et al. [38] tested the safety and feasibility of minocycline in treatment of acute traumatic brain injury patients and found that minocycline was safe for moderate to severe TBI at a dose twice that as recommended for treatment of infection. A Phase II minocycline trial was conducted to examine its effect against human spinal cord injury and found that HO1 and neurofilament heavy chain in the cerebrospinal fluid were changed by minocycline treatment [39]. Sangobowale et al. [40] treated mice with closed head injury with minocycline plus N-acetylcysteine (NAC) 12 h later and revealed that MINO plus NAC or MINO alone when first dosed 12 h after CHI increased myelin content using similar mechanisms seen when first dosed 1 h after closed head injury, suggesting that drugs protect oligodendrocytes with a clinically useful therapeutic time window. Minocycline could also attenuate TBI injury in post-natal Day 17 rats [41]. However, there are also some confusing results regarding the protective effect of MINO against TBI. Scott et al. [26] found out that minocycline reduced chronic microglial activation Table 1 Iron concentration in the brain of rats (μg/g tissue).
Cortex Hippocampus Whole brain
Control
Sham
TBI + Vehicle
TBI + MINO
MINO
18.66 ± 3.18 40.28 ± 2.41 14.96 ± 2.58
17.89 ± 2.59 42.56 ± 3.69 15.45 ± 2.86
35.87 ± 3.17# 69.45 ± 4.99# 14.55 ± 3.59
25.45 ± 2.94* 51.85 ± 3.46* 13.54 ± 3.22
21.45 ± 2.31 42.87 ± 3.28 14.79 ± 3.39
The iron concentration in the cortex, hippocampus and whole brain of rats was measured. Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and minocycline injection; MINO: minocycline injection only. TBI: traumatic brain injury; MINO: minocycline. #: p < 0.05 compared to Sham. *: p < 0.05 compared to TBI + Vehicle. N = 10 per group. 7
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Fig. 6. Effects of minocycline on the expression of iron metabolism proteins in the hippocampus and cortex. Protein levels of ferritin (a), TfR1 (b), DMT1 (b), FPN1 (c), and hepcidin (d). Control: no treatment; Sham: sham surgery; TBI + Vehicle: TBI surgery and saline injection; TBI + MINO: TBI surgery and 20 mg/kg minocycline injection; MINO: 20 mg/kg minocycline injection only. TfR1: transferrin receptor 1; DMT1: divalent metal transporter 1; FPN1: ferroportin 1; TBI: traumatic brain injury; MINO: minocycline; #P < 0.05 compared to Sham; *P < 0.05 compared to TBI + Vehicle; n = 6 per group.
in these organs. When iron in the blood increases, transferrin receptor (TfR)-mediated iron absorption in liver also increases, which leads to increased hepcidin synthesis and secretion. As the hepcidin concentration in the blood increases, more hepcidin arrives at the target organ and then suppresses the iron absorption in the small intestine. Meanwhile, it increases the iron uptake by liver cells, bone marrow cells and macrophages, thereby increasing iron storage and utilization. Through this hepcidin-mediated pathway, blood iron concentrations will return to normal levels. Recent studies have confirmed that the brain itself is capable of expressing hepcidin and hepcidin is widely distributed in the brain [46–48]. It was found that hepcidin was expressed in endothelium and pericytes of blood vessels, glial cells, olfactory bulb, sub-ventricular zone and dentate gyrus. It was also shown that hepcidin co-localised with FPN in ependymal cells of the subventricular zone and in the corpus callosum. A significant increase in the expression of iron uptake (TfR1 and DMT1) and release (Fpn1) proteins was found in astrocytes [49]. Previous studies have found Fpn1 in neurons, astrocytes, and oligodendrocytes, which indicated that Fpn1 might be essential in iron efflux. Tf in the brain can be produced in oligodendrocytes or epithelial cells of the choroid plexus and Tf-Fe can be taken up by neurons and astrocytes via a TfR1-mediated process [50]. Our results showed that FPN1 was significantly decreased in the TBI + Vehicle group, but recovered in the TBI + MINO group. By contrast, the protein levels of hepcidin were significantly increased in the TBI + Vehicle group, but decreased in the TBI + MINO group. This is probably a result of the increase in the iron concentration, which leads to increased hepcidin synthesis and secretion. These results indicate that minocycline may restore the expression of FPN1 by inhibiting the overexpression of hepcidin. Furthermore, it is interesting to note that minocycline treatment reduced brain iron accumulation and suppressed iron metabolizing protein upregulation, but did not alter the normal level of iron or iron metabolizing proteins in healthy rats. Our study has showed that minocycline has strong iron-chelating activity. It is possible that the difference between the state of iron of TBI rats and healthy rats may lead to the iron-chelating activity of minocycline; therefore minocycline treatment reduced brain iron accumulation in TBI rats, but did not alter the normal level of iron in sham treated rats. Regarding the iron metabolizing proteins, it is possible that the change of iron metabolizing proteins is a response to the change of iron level in the brain. Minocycline may don't directly change the expression of
blood cells in the wound can be dissolved, which releases a large amount of heme and iron ions, causing neurotoxicity, brain atrophy, and neurological impairment [43], which may be the reason that the local iron concentration were increased, but the overall iron concentration did not increase to a significant level. The iron concentrations in serum, however, were not affected by minocycline, indicating that the iron-reducing effect of minocycline was limited locally. The in vitro study showed that minocycline could directly bind Fe2+ or Fe3+, indicating that directly reducing the iron concentration may be a mechanism of minocycline. In the present study, a significant increase in ferritin expression was observed in the TBI + Vehicle group, suggesting that ferritin was upregulated in the brain tissue to increase iron ion storage to avoid damage from iron ions. Treatment with minocycline significantly decreased ferritin levels in the brain tissue, probably because it significantly reduced the level of iron. The fact that minocycline treatment alone did not change the ferritin level suggests that minocycline did not directly regulate ferritin expression. The Tf/TfR pathway is the main route of iron transport. Xi et al. [13] found that ferritin was upregulated after experimental intracerebral hemorrhage, and the uptake of iron by cells increased, resulting in neurotoxicity. The level of TfR1 of the TBI + Vehicle group in the hippocampus and cortex was significantly higher than that in the Sham group. Its expression in the TBI + MINO group, however, was significantly decreased compared to that in the TBI + Vehicle group. This result suggests that ferritin is also upregulated after TBI, causing neurotoxicity, but treatment with minocycline can inhibit its increase, thus inhibiting TBI-induced neurotoxicity. However, the expression of another important iron metabolism protein, DMT1, was not affected by the TBI model or minocycline, indicating that DMT1 may not be involved. To further investigate the mechanism by which minocycline regulates iron metabolism, we examined the expression levels of FPN1 and hepcidin. Studies have shown that FPN1 is essential for iron export from neurons [44]. Hepcidin is a major regulator of the activity of FPN1, which is mainly expressed in astrocytes [45]. Increased levels of hepcidin can lead to internalization and degradation of FPN1 by binding to its evolutionarily conserved hepcidin-binding domain, which eventually causes iron accumulation in neurons [44]. Hepcidin is mainly synthesized in the liver and then secreted into the blood. It is transported to its main target organs, including the small intestine, liver, bone marrow cells and macrophages, and controls the iron metabolism 8
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these iron metabolizing proteins, but can alter their expression by chelating excess iron in the TBI. As a result, it inhibited the over expression of iron metabolizing proteins in the TBI rats, but did not change the normal level of iron metabolizing proteins in the sham treated rats. In conclusion, the present study shows that minocycline significantly attenuated the neurological impairment caused by TBI, and increased neuronal viability. Minocycline reduced the iron concentrations in CSF and brain tissues (cortex and hippocampus) and had ironchelating activity. Minocycline also inhibited the overexpression of ferritin and TfR1 in the hippocampus and cortex, but did not affect the expression of DMT1. Minocycline restored the expression of FPN1 by decreasing the expression of hepcidin. Taken together, these results suggest that minocycline attenuates neurological impairment caused by TBI and regulates iron metabolism.
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CRediT authorship contribution statement Lijun Zhang: Methodology, Validation, Investigation, Funding acquisition, Writing - original draft. Hong Xiao: Formal analysis, Writing - review & editing. Xing Yu: Resources, Data curation. Yongbing Deng: Conceptualization, Funding acquisition. Declaration of competing interest There is no conflict of interest. Acknowledgements This work was supported by Research on Basic Science and Advanced Techniques in Chongqing (cstc2015jcyjA10098), Science and Technology Project of Taizhou, Zhejiang, China (1901ky57) and Research on Basic Science and Advanced Techniques of Yuzhong District, Chongqing (20170139). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.abb.2020.108302. References [1] M.A. Vella, M.L. Crandall, M.B. Patel, Acute management of traumatic brain injury, Surg. Clin. North Am. 97 (2017) 1015–1030. [2] M. Galgano, G. Toshkezi, X. Qiu, T. Russell, L. Chin, L.R. Zhao, Traumatic brain injury: current treatment strategies and future endeavors, Cell Transplant. 26 (2017) 1118–1130. [3] A.I. Maas, N. Stocchetti, R. Bullock, Moderate and severe traumatic brain injury in adults, Lancet Neurol. 7 (2008) 728–741. [4] X. Wu, J. Hu, L. Zhuo, C. Fu, G. Hui, Y. Wang, W. Yang, L. Teng, S. Lu, G. Xu, Epidemiology of traumatic brain injury in eastern China, 2004: a prospective large case study, J. Trauma 64 (2008) 1313–1319. [5] D.K. Menon, Unique challenges in clinical trials in traumatic brain injury, Crit. Care Med. 37 (2009) S129–S135. [6] S. Rooker, S. Jander, J. Van Reempts, G. Stoll, P.G. Jorens, M. Borgers, J. Verlooy, Spatiotemporal pattern of neuroinflammation after impact-acceleration closed head injury in the rat, Mediat. Inflamm. 1 (2006) 90123. [7] H.J. Thompson, N. Marklund, D.G. LeBold, D.M. Morales, C.A. Keck, M. Vinson, N.C. Royo, R. Grundy, T.K. McIntosh, Tissue sparing and functional recovery following experimental traumatic brain injury is provided by treatment with an antimyelin-associated glycoprotein antibody, Eur. J. Neurosci. 24 (2006) 3063–3072. [8] S. Altamura, M.U. Muckenthaler, Iron toxicity in diseases of aging: Alzheimer's disease, Parkinson's disease and atherosclerosis, J. Alzheim. Dis. 16 (2009) 879–895. [9] Y. Ke, Z.M. Qian, Brain iron metabolism: neurobiology and neurochemistry, Prog. Neurobiol. 83 (2007) 149–173. [10] D.A. Long, K. Ghosh, A.N. Moore, C.E. Dixon, P.K. Dash, Deferoxamine improves spatial memory performance following experimental brain injury in rats, Brain Res. 717 (1996) 109–117. [11] J. Zhao, Z. Chen, G. Xi, R.F. Keep, Y. Hua, Deferoxamine attenuates acute hydrocephalus after traumatic brain injury in rats, Transl. Stroke Res. 5 (2014) 586–594. [12] Y. Hua, T. Nakamura, R.F. Keep, J. Wu, T. Schallert, J.T. Hoff, G. Xi, Long-term effects of experimental intracerebral hemorrhage: the role of iron, J. Neurosurg.
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