The interleukin-1 receptor antagonist (IL-1RA) is a potential stroke treatment candidate. Intranasal delivery is a novel method thereby a therapeutic protein can be penetrated into the brain parenchyma by bypassing the blood-brain barrier. Thus, this study tested whether intranasal IL-1RA can provide neuroprotection and brain penetration in transient cerebral ischemia. In male Sprague-Dawley rats, focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 1 h. The rats simultaneously received 50 mg/kg human IL-1RA through the intranasal (IN group) or intraperitoneal route (IP group). The other rats were given 0.5 mL/kg normal saline (EC group). Neurobehavioral function, infarct size, and the concentration of the administered human IL-1RA in the brain tissue were assessed. In addition, the cellular distribution of intranasal IL-1RA in the brain and its effect on proinflammatory cytokines expression were evaluated. Intranasal IL-1RA improved neurological deficit and reduced infarct size until 7 days after MCAO (
Stroke is a major cause of death and disability in adults worldwide. Although the etiology of stroke may be ischemia or hemorrhage, ischemic stroke accounts for about 80% of all stroke cases (Chalela
Inflammation is a major part of the pathophysiology of stroke (Amantea
The blood-brain barrier (BBB) is known to be a major barrier against the delivery of pharmacologically relevant quantities of therapeutic proteins to the brain. IL-1RA is a large (17 kDa) hydrophilic peptide. Thus, the penetration of peripherally administered IL-1RA into the brain was reported to be limited (Gutierrez
This experiment protocol was approved by the Yonsei University Animal Care and Use Committee (protocol number: 2012-0235; Seoul, Korea) and was in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. Male Sprague-Dawley rats aged 8–10 weeks, weighing 280–320 g, were obtained from Orientbio Inc. (Seongnam, Korea) and used for this study. The rats were maintained under a 12-h light-dark cycle and allowed free access to food and water before and after surgery.
Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery (MCA). The experimental MCA occlusion (MCAO) was performed as previously described (Belayev
The rats were randomly divided into 3 groups. All of the rats underwent MCAO/reperfusion as described earlier. The IN group intranasally received 50 mg/kg human IL-1RA (Kineret, Amgen Manufacturing, Ltd., Thousand Oaks, CA, USA). Intra-nasal administration of 0.5 mL/kg Kineret (undiluted solution, approximately 100 μL) was performed in 8- to 10-μL drops by using a pipette, treating each nare every 5 min for 60 min from the start of MCAO. In addition, 0.5 mL/kg 0.9% saline was administered intraperitoneally at the start of MCAO. The IP group received 50 mg/kg IL-1RA intraperitoneally at the start of MCAO and the same volume of 0.9% saline intranasally in the same manner as did the IN group. The EC group received 0.5 mL/kg 0.9% saline through both the intranasal and intraperitoneal routes.
The administration dose of human IL-1RA was determined based on previous studies. The systemic administration of 100 mg/kg human IL-1RA showed neuroprotective effects in rat models of cerebral ischemia when given at the beginning of ischemia (Garcia
Neurological deficits were assessed before and 1, 2, 4, and 7 days after MCAO. Two examiners who were blinded to the treatment conditions consecutively and independently performed the neurological examinations of the animals. Adherence to a predetermined time excluded behavioral changes based on the circadian rhythm. The neurological examination consisted of 6 tests developed and described by Garcia
The rota-rod test was used to assess the recovery of impaired motor function after MCAO. This accelerating rota-rod test (ENV-577, Med Associates Inc., Georgia, VT, USA) was conducted as described by Hunter
On the seventh day after MCAO, the animals were anesthetized and decapitated. The brains were quickly isolated and sectioned into 2-mm-thick serial coronal slices. The brain slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma Aldrich, St Louis, MO, USA) in the dark at 37°C for 30 min and fixed with 4% paraformaldehyde (PFA, Sigma Aldrich) overnight. The posterior surface of each slice was photographed and analyzed by using a computer-assisted image analysis system (Optimas ver 6.1, Optimas, Bothell, WA, USA). The lesion volume was calculated by multiplying the area by the thickness of the slices. We adopted a previously described method to eliminate the contribution of edema to the ischemic lesion and to correct for the individual difference in brain volumes by using the percentage of infarct volume in the ipsilateral hemisphere volume (Belayev
Serum and brain tissues, including the striatum and cortex, were obtained during appointment and were stored in aliquots at −80°C until use for different assays. Serum levels of human IL-1RA were measured by using high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits (Quantikine ELISA, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. To measure the human IL-1RA level in brain tissue, total proteins of brain tissues, including the striatum and cortex, were extracted by using a tissue protein extraction reagent (T-PER Tissue Protein Extraction Reagent, Thermo Scientific, Waltham, MA, USA). In brief, the weight of the striatal and cortical tissues was measured, and the reagent was added (20-μL reagent per 1-mg tissue). Inhibitor cocktail (100X Halt protease and phosphate inhibitor cocktail, Thermo 1861281, Thermo Scientific) were also added to the reagent for protection of intact active cellular proteins from degradation, and the striatal and cortical tissues were then homogenized. After centrifugation, the supernatant was collected. Protein concentration was determined by using the BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s methods. To calculate the protein yield in a maximum of 20,000 pg/mL, we measured the sample area by using the manufacturer’s instruction standard. The human IL-1RA levels in brain tissue lysate were assayed in high sensitivity ELISA kits (Quantikine ELISA, R&D Systems) according to the manufacturer’s instructions. Samples were added at 100 μL per plate and incubated for 2 h at room temperature. After washing with a wash buffer, 200 μL of human IL-1RA conjugates was added to each sample and incubated for 2 h. After reaction, the wavelength was read at 450 nm by using a microplate reader.
Total RNA was prepared from the cortex of the ipsilateral hemisphere by using an RNeasy Mini Kit (Qiagen, Austin, Texas, USA) according to the manufacturer’s directions. Briefly, recommended amounts of tissue (25 mg) were placed in the appropriate lysis buffer supplied with the kit (add 10 μL of β-mercaptoethanol to 1 mL of lysis buffer). At this point, the manufacturer’s protocol was followed. The RNA was eluted with 30–50 μL of RNase-free H2O. The samples were immediately aliquoted and stored at −80°C. RNA was quantified by measuring A260 absorbance in NanoDrop ND-1000 (Thermo Scientific). Purity was assessed by calculating the A260/A280 ratio. cDNA was synthesized with 1 μg of total RNA from each sample by using a Maxime Reverse Transcriptase (RT) PreMix kit (OligodT Primer, iNtRON Biotechnology, Inc., Seongnam, Korea). The diluted cDNA was amplified with SYBR Green Polymerase Chain Reaction (PCR) Master Mix (Applied Bio-systems, Foster City, CA, USA) in a final volume of 50 μL. The PCR was performed by using an ABI prism 7500 sequence detector (Applied Biosystems). The PCR program consisted of initial denaturation at 95°C for 10 s and 40 cycles at 95°C for 5 s, 60°C for 34 s, 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The primers for b-actin (forward, 5′-GGCATCCTGACCCTGA-AGTA-3′ and reverse, 5′-GGGGTGTTGAAGGTCTCAAA-3′), IL-1β (forward, 5′-CACCTCTCAAGCAGAGCACAG-3′ and reverse, 5′-GGGTTCCATGGTGAAGTCAAC-3′), TNF-α (forward, 5′-CAGGAGAAAGTCAGCCTCCT-3′ and reverse, 5′-TCATA-CCAGGGCTTGAGCTCA-3′), and IL-6 (forward, 5′-CGAAAGT-CAACTCCATCTGCC-3′ and reverse, 5′-GGCAACTGGCTG-GAAGTCTCT-3′) were purchased from Bioneer Inc (Seongnam, Korea). The cycling threshold (Ct) values of IL-1β, IL-6, and TNF-α were normalized to Ct values of b-actin. All the samples were run in triplicate, and the animals that underwent sham surgery without MCAO served as controls.
Brain tissue samples were obtained at 6 h after MCAO and fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS). After fixation, the isolated brain tissues were soaked in 30% sucrose at 4°C overnight. For cryosection, the samples were embedded in optimum cutting temperature compound (Tissue-Tek, Sakura, Japan) and frozen at −80°C. The brain tissues were sectioned coronally at a 20-μm thickness by using a cryotome (Leica Instruments GmbH, Nussloch, Germany) and placed on Muto silane-coated microscope slides (Muto Pure Chemicals, Tokyo, Japan). For immunohistochemistry of NeuN, Iba-1, IL-1β, and IL-1ra, the slides were defrosted and immersed in 0.3% Triton X-100 for 1 h for antigen retrieval and washed with PBS 3 times for 5 min. Then, the samples were incubated with EtOH at −20°C for 10 min and dried for 30 sec. Bovine serum albumin (BSA) solution (5%) was used for the blocking process. Tissue sections were incubated overnight at 4°C with primary antibodies, diluted in 2% BSA with PBS such as monoclonal mouse anti-NeuN (1:50; Millipore, Bedford, MA, USA), monoclonal mouse anti-Iba-1 (1:50; Abcam, Cambridge, UK), polyclonal rabbit anti-IL-1β (1:50, Abcam), and polyclonal goat anti-IL-1ra (R&D Systems). The next day, fluorescein isothiocyanate-conjugated anti-mouse IgG (1:5,000; Millipore), rhodamine-conjugated anti-goat IgG (1:5,000; Millipore), rhodamine-conjugated anti-rabbit IgG antibodies (1: 5,000; Millipore) were incubated at room temperature for 1 h as secondary antibodies. The slides were washed with PBS. 4′6-Diamidino-2-phenylindole was used for counterstaining, and slides were covered with VECTASHIELD mounting medium (Vecta Laboratories, CA, USA). The tissue sections were observed under an LSM700 confocal laser scanning microscope (Carl Zeiss, Thornwood, NY, USA).
All data were presented as mean (standard error of the mean [SEM]). The 2 groups were compared by performing an independent 2-tailed
In order to evaluate the effects of intranasal IL-1RA on the neurological outcome of transient focal cerebral ischemia, neurobehavioral function, infarct, and edema volume were assessed. Two rats in the EC group and one in the IP group died before the completion of the assessments. The comparisons for infarct and edema size were performed by using one-way analysis of variance with the Bonferroni post hoc test (Fig. 1). On the seventh day after MCAO, the IN group had significantly lesser infarct volume than the EC group (
The neurological score before MCAO was 18 in all the experimental rats because no neurological deficit was present. By the seventh day after MCAO, the IN group showed significantly greater neurological scores than the EC and IP groups (
Human IL-1RA concentrations in brain parenchyma, including the cortex and striatum, and serum were measured at 1, 3, 6, and 24 h after MCAO. Fig. 3 shows the human IL-1RA concentrations in serum and brain tissue. After intranasal administration, human IL-1RA already reached significant concentrations in both of the striatum and cortex at the first sampling time (1 h after MCAO; Fig. 3A, 3B). In the striatum, the IL-1RA concentration at 1 h after MCAO was highest (11,265 ± 2,105 pg/mL) among the measurement time points. On the other hand, the IL-1RA concentration in the cortex was the highest at 3 h after MCAO (10,130 ± 750 pg/mL). After intraperitoneal administration, the IL-1RA concentration was highest at 3 h after MCAO in both the striatum (2,369 ± 1,097 pg/mL) and cortex (3,682 ± 1,863 pg/mL). In the all measurement time points, the IL-1RA concentrations were significantly greater in the IN group than in the IP group (by independent 2-tailed
Fig. 3C depicts the serum levels of human IL-1RA. The IL-1RA levels at 1 h after MCAO were highest in both groups (IN group: 222,274 ± 47,209 pg/mL [222 ng/mL]; IP group: 31,661,574 ± 2,403,415 pg/mL [31,662 ng/mL]). At all the measurement time points, the serum concentrations of IL-1RA were significantly higher in the IP group than in the IN group (by independent 2-tailed
According to the manufacturer, the anti-human IL-1RA anti-body used in the ELISA in this study shows no cross-reactivity with rat IL-1RA. We also confirmed that IL-1RA was not detected in the EC group over all the measurement time points.
Cellular distribution of intranasal IL-1RA was assessed by using immunohistochemistry 6 h after MCAO. Fig. 4 shows the immunoreactivity of IL-1RA, which coincides with the NeuN (Fig. 4B) and Iba1 staining (Fig. 4C) in the ipsilateral striatum and cortex. The EC group rarely had IL-1RA staining in both the striatum and cortex. On the other hand, the IN group demonstrated strong immunoreactivity of IL-RA, which was largely colocalized with NeuN (Fig. 4A) and Iba-1 (Fig. 4B). According to the manufacturer of the anti-human IL-1RA antibody used in the immunohistochemistry, cross-reactivity with rat IL-1RA has not been reported yet (only less than 15% cross-reactivity with mouse was described). However, minimal immunoreactivity was observed in the EC group.
Fig. 5 exhibits the IL-1β expression 6 h after MCAO in the cortex and striatum. The EC group had greater IL-1β immuno-reactivity, which was colocalized with NeuN (Fig. 5A) and Iba1 (Fig. 5B) when compared with the IN group, which imposed that intranasal IL-1RA suppressed the IL-1β expression.
The effects of intranasal IL-1RA on the mRNA levels of IL-1β and TNF-α after transient focal cerebral ischemia were evaluated by using semiquantitative RT-PCR. The values were expressed as a ratio in comparison with the sham control. As shown in Fig. 6, the IN group had significantly lower mRNA levels of IL-1β and TNF-α than the EC group at 6 h after MCAO (by independent 2-tailed
The present study demonstrated that intranasal delivery of IL-1RA significantly reduced the size of infarct lesion and alleviated neurological impairment by 7 days after transient focal cerebral ischemia. However, systemic administration of IL-1RA neither significantly decreased the infarct size nor improved neurological impairment. The administered human IL-1RA concentrations of brain tissue were significantly higher in the intranasal than in the systemic administration at all measurement points for 24 h after cerebral ischemia. On the other hand, in the serum, the administered IL-1RA was detected significantly less in the intranasal than in the systemic administration at every measurement time point. Finally, our results showed that the intranasally administered IL-1RA was deposited in neurons and activated microglia, and suppressed inflammatory cytokines after cerebral ischemia. Therefore, the superior neuroprotective efficacy of intranasal administration to systemic administration may have resulted from the greater delivery of IL-1RA into brain tissues, which can suppress neuroinflammation after transient focal cerebral ischemia.
Previous experimental studies already demonstrated the efficacy of IL-1RA in cerebral ischemia model 2 decades ago (Martin
Until recently, the intranasal administration of various therapeutic proteins has been investigated not only in stroke models (Fletcher
As a response to ischemic brain injury, cytokines are secreted from immune cells to trigger an inflammatory response. The cytokines that are produced in the early phase after cerebral ischemia are reported to be IL-1β and TNF-α (Lambertsen
There is a concern that successful application of intranasal administration in rodents may not be reproduced in humans (Grassin Delyle
In conclusion, intranasal administration of IL-1RA was a more efficient route to reach brain parenchyma and showed superior efficacy in neuroprotection against transient cerebral ischemia in the rats when compared with systemic administration. Therefore, intranasal delivery of IL-1RA should be investigated further as a potential therapeutic option for ischemic stroke.
This study was financially supported by the “Dongwha Holdings” Faculty Research Assistance Program of Yonsei University College of Medicine for 2013 (No. 6-2013-0067), the Basic Science Research Program through the National Research Foundation of Korea (NRF) that was funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A1A1002001) to J.H.L. and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2014R1A2A2A01007289) to K.B.-N.