Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 avian influenza virus (AIV).

B2 haplotype major histocompatibility complex (MHC) has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown, and the presenting peptide is rarely identified. Here, we identified its peptide-binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) in vitro using Random Peptide Library-based MHC I LC-MS/MS analysis. To further clarify the structure basis of motif, we determined the crystal structure of the BF2∗02:01-PB2552-560 complex at 1.9 Å resolution. We found that BF2∗02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2∗02:01 than BF2∗04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterward, we explored the H9N2 avian influenza virus (AIV)-induced cellular immune response in B2 haplotype chickens in vivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, interferon-γ, NK lysin, and poly-(ADP-ribose) polymerase in peripheral blood mononuclear cells were significantly increased in defending against H9N2 AIV infection. Especially, we selected 425 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot interferon-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.

Analysis of H5N8 influenza virus infection in chicken with mApple reporter genes in vivo and in vitro.

H5N8 highly pathogenic avian influenza virus (HPAIV) has caused huge losses to the global poultry industry and critically threatens public health. Chickens are the important host for the transmission. However, the distribution of H5N8 avian influenza virus (AIV) in chicken and the infected cell types are limitedly studied. Therefore, in this study, we detected viral replication and infection by generating recombinant H5N8 AIV expressing an easily tracked mApple fluorescent reporter. The results showed that recombinant viruses passaged four times in chicken embryos successfully expressed mApple proteins detected by fluorescence microscopy and WB, which verified that the constructed recombinant viruses were stable. Compared to parental virus, although recombinant virus attenuated for replication in MDCK cells, it can still replicate effectively, and form visible plaques. Importantly, the experiments on infection of chicken PBMCs in vitro showed a strong correlation between mApple positivity rate and NP positivity rate (r = 0.7594, P =0.0176), demonstrating that mApple reporter could be used as an indicator to accurately reflect AIV infection. Then we infected monocytes/macrophages in PBMCs in vitro and detected the mApple positive percentage was 55.1%-80.4%, which confirmed the chicken primary monocytic/macrophages are important target cells for avian influenza virus infection. In chicken, compared with parental virus, the recombinant virus-infected chickens had lower viral titers in oropharyngeal cloacal and organs, but it can cause significant pathogenicity in chicken and the mortality rate was approximately 66%. In addition, the results of bioluminescent imaging showed that the fluorescence in the lungs was strongest at 5 days post-infection (DPI). Finally, we discovered the mApple positive expression in chicken lung immune cells (CD45+ cells), especially some T cells (CD4 and CD8 T cells) also carrying mApple, which indicates that the H5N8 AIV showed a tropism for immune cells including chicken T cells causing potentially aggressive against cellular immunity. We have provided a simple visualization for further exploration of H5N8 AIV infected chicken immune cells, which contributes to further understanding pathogenic mechanism of H5N8 AIV infection in chicken.

Dissection of key factors correlating with H5N1 avian influenza virus driven inflammatory lung injury of chicken identified by single-cell analysis.

Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages (clusters 0, 1, and 14) with massive viral replication, pro-inflammatory cytokines (IFN-ß, IL1ß, IL6 and IL8), and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection.

Duck CD8+ T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro.

Domestic ducks are the important host for H5N1 highly pathogenic avian influenza virus (HPAIV) infection and epidemiology, but little is known about the duck T cell response to H5N1 AIV infection. In infection experiments of mallard ducks, we detected significantly increased CD8+ cells and augmented expression of cytotoxicity-associated genes, including granzyme A and IFN-γ, in PBMCs from 5 to 9 d postinfection when the virus shedding was clearly decreased, which suggested the importance of the duck cytotoxic T cell response in eliminating H5N1 infection in vivo. Intriguingly, we found that a CD8high+ population of PBMCs was clearly upregulated in infected ducks from 7 to 9 d postinfection compared with uninfected ducks. Next, we used Smart-Seq2 technology to investigate the heterogeneity and transcriptional differences of the duck CD8+ cells. Thus, CD8high+ cells were likely to be more responsive to H5N1 AIV infection, based on the high level of expression of genes involved in T cell responses, activation, and proliferation, including MALT1, ITK, LCK, CD3E, CD247, CFLAR, IL-18R1, and IL-18RAP. More importantly, we have also successfully cultured H5N1 AIV-specific duck T cells in vitro, to our knowledge, for the first time, and demonstrated that the CD8high+ population was increased with the duck T cell activation and response in vitro, which was consistent with results in vivo. Thus, the duck CD8high+ cells represent a potentially effective immune response to H5N1 AIV infection in vivo and in vitro. These findings provide novel insights and direction for developing effective H5N1 AIV vaccines.

Genomic Sequence and Pathogenicity of the Chicken Anemia Virus Isolated From Chicken in Yunnan Province, China.

Chicken anemia virus (CAV), which has been reported in many countries, causes severe anemia and immunosuppression in chickens. In this study, a CAV strain YN04 belonging to genotype A was first identified from infected chickens in Yunnan province, China. Moreover, the animal infection experiments further confirmed that the strain YN04 is a highly pathogenic strain, which can cause 86.67% mortality in chickens in the infection group. The mean death time of infected chickens was 13.1 days post infection (dpi). CAV infection induced severe anemia with significant decrease in packed cell volume (PCV), and serious atrophy and lesion of thymus and bursa with high viral load at 14 dpi. Besides, CAV infection caused a sharp decrease in chicken body weight and immune organ indices including the ratio of thymus or bursa to body weight at 21 dpi, which displayed the potential immunosuppression state at this stage. These findings enrich the epidemiological data on CAV and may provide information for preventing its further spread in Yunnan province, China.

Endogenous retroviruses transcriptomes in response to four avian pathogenic microorganisms infection in chicken.

The impact of Endogenous retroviruses (ERVs) on chicken disease is not well understood. Here, we systematically identified 436 relatively complete ChERVs from the chicken genome. Subsequently, ChERV transcriptomes were analyzed in chicken after subgroup J avian leukosis virus (ALV-J), avian influenza virus (AIV), Marek's disease virus (MDV) and avian pathogenic Escherichia coli (APEC) infection. We found that about 50%-68% of ChERVs were transcriptionally active in infected and uninfected-samples, although the abundance of most ChERVs is relatively low. Moreover, compared to uninfected-samples, 49, 18, 66 and 17 ChERVs were significantly differentially expressed in ALV-J, AIV, MDV and APEC infected-samples, respectively. These findings may be of significance for understanding the role and function of ChERVs to response the pathogenic microorganism infection.

Chicken Peripheral Blood Mononuclear Cells Response to Avian Leukosis Virus Subgroup J Infection Assessed by Single-Cell RNA Sequencing.

Chicken peripheral blood mononuclear cells (PBMCs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Here we performed single-cell RNA sequencing (scRNA-seq) of PBMCs in Avian leukosis virus subgroup J (ALV-J) infected and control chickens at 21 days post infection (DPI) to determine chicken PBMCs subsets and their specific molecular and cellular characteristics. Eight cell populations and their potential marker genes were identified in PBMCs. T cell populations had the strongest response to (ALV-J) infection, based on the detection of the largest number of differentially expressed genes (DEGs), and could be further grouped into four subsets: activated CD4+ T cells, Th1-like cells, Th2-like cells, and cytotoxic CD8+ T cells. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection activated CD4+ T cell was probably inclined to differentiate into Th1-like cells. Compared to the control PBMCs, ALV-J infection also had an obvious impact on PBMCs composition. B cells showed inconspicuous response and their numbers decreased in PBMCs from ALV-J infected chicken. Proportions of cytotoxic Th1-like cells and CD8+ T cells increased in the T cell population of PBMCs from ALV-J infected chicken, which were potentially key mitigating effectors against ALV-J infection. More importantly, our results provide a rich resource of gene expression profiles of chicken PBMCs subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.

The Transcriptional Differences of Avian CD4+CD8+ Double-Positive T Cells and CD8+ T Cells From Peripheral Blood of ALV-J Infected Chickens Revealed by Smart-Seq2.

It is well known that chicken CD8+ T cell response is vital to clearing viral infections. However, the differences between T cell subsets expressing CD8 receptors in chicken peripheral blood mononuclear cells (PBMCs) have not been compared. Herein, we used Smart-Seq2 scRNA-seq technology to characterize the difference of chicken CD8high+, CD8high αα+, CD8high αß+, CD8medium+, and CD4+CD8low+ T cell subsets from PBMCs of avian leukosis virus subgroup J (ALV-J)-infected chickens. Weighted gene co-expression network analysis (WGCNA) and Trend analysis revealed that genes enriched in the "Cytokine-cytokine receptor interaction" pathway were most highly expressed in the CD8high αα+ T cell population, especially T cell activation or response-related genes including CD40LG, IL2RA, IL2RB, IL17A, IL1R1, TNFRSF25, and TNFRSF11, suggesting that CD8high αα+ T cells rather than other CD8 subpopulations were more responsive to ALV-J infections. On the other hand, genes involved in the "FoxO signaling pathway" and "TGF-beta signaling pathway" were most highly expressed in the CD4+CD8low+ (CD8low+) T cell population and the function of CD4+CD8low+ T cells may play roles in negatively regulating the functions of T cells based on the high expression of CCND1, ROCK1, FOXO1, FOXO3, TNFRSF18, and TNFRSF21. The selected gene expressions in CD8+ T cells and CD4+CD8low+ double-positive T cells confirmed by qRT-PCR matched the Smart-Seq2 data, indicating the reliability of the smart-seq results. The high expressions of Granzyme K, Granzyme A, and CCL5 indicated the positive response of CD8+ T cells. Conversely, CD4+CD8+ T cells may have the suppressor activity based on the low expression of activation molecules but high expression of T cell activity suppressor genes. These findings verified the heterogeneity and transcriptional differences of T cells expressing CD8 receptors in chicken PBMCs.

Rapid detection of SARS-CoV-2, replicating or non-replicating, using RT-PCR.

To identify animals susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection or to determine whether SARS-CoV-2 contaminated meat is from a SARS-CoV-2-infected animal, a convenient and safe method was developed for rapid detection of SARS-CoV-2 in a replicating or non-replicating status in samples using reverse transcriptase-polymerase chain reaction (RT-PCR). This strategy can also be applied to develop assays for the detection of other viruses, either replicating or not.

Comparative analysis of key immune protection factors in H9N2 avian influenza viruses infected and immunized specific pathogen-free chicken.

H9N2 avian influenza viruses (AIV) continue to circulate in vaccinated chicken flocks in China, which prompted us to investigate the differential immune protection factors induced by H9N2 AIV infection and immunization for analyzing the reason of protection deficiency of H9N2 AIV inactivated vaccine. In this study, we firstly explored virus-induced optimal immune responses in chicken after H9N2 AIV infection. And, we found that H9N2 hemagglutination inhibition (HI) antibody level, antiviral interferon-stimulated genes including 2',5'-oligoadenylate synthetase-like and myxovirus resistance 1, CD8+ T cell response in peripheral blood lymphocytes (PBL) accompanied by the cytotoxicity-associated genes, including poly (ADP-ribose) polymerase and IFN-r play important roles in defending against H9N2 infection. Besides, we observed that vaccine immunization triggered the similar H9N2 HI antibody level as viral infection, the increase of CD4+ T cell percentage instead of CD8+ T cell percentage in PBL. Moreover, we further made a comparative analysis of immune-related gene expression profile in PBL and lung after H9N2 AIV infection and immunization, respectively. The results showed that vaccine immunization contributed to the up-regulation of Th2 cytokine. But the deficiency of cytotoxicity-associated genes induced by H9N2 AIV inactivated vaccine may be the potential key reason of protection deficiency. These findings provide evidence and direction for developing effective H9N2 AIV vaccines.

Design, Characterization, and Application of a pH-Triggered In Situ Gel for Ocular Delivery of Vinpocetine.

We developed a pH-triggered in situ gel (ISG) for ocular delivery of vinpocetine to achieve systemic absorption and a brain-targeting effect in rats. Carbopol acted as a gelling agent combined with hydroxypropyl methylcellulose (HPMC) as a viscosity-enhancing agent. The concentration of Carbopol (0.2%, w/v) and HPMC (1.5%, w/v) was optimized for the ISG system. The optimized formulation was evaluated for studies on release in vitro, rheology, differential scanning calorimetry, ocular irritation, residence time, and in vivo pharmacokinetics. The vinpocetine ISG stayed longer in rabbit eyes than vinpocetine ointment. In vivo pharmacokinetics showed that compared with vinpocetine ointment, vinpocetine ISG attained a peak plasma concentration and area under the curve that was 1-2 folds greater in rat plasma. The Drug Targeting Index (DTI) was 1.06 and 1.26 for vinpocetine ointment and vinpocetine ISG, respectively, after ocular administration, showing that vinpocetine ISG had better distribution in rat brain. These results revealed that a pH-triggered ISG system via ocular administration could be an alternative approach compared with traditional ophthalmic formulations.

Comparative Pharmacokinetics of Nimodipine in Rat Plasma and Tissues Following Intraocular, Intragastric, and Intravenous Administration.

We investigated the pharmacokinetics of nimodipine (NMD) in rats plasma and tissues following intraocular (io), intragastric (ig), and intravenous (iv) administration at doses of 5.0 mg/kg io and iv and 10.0 mg/kg ig. After a single dose of NMD, plasma, heart, liver, spleen, lung, kidney, and brain samples were collected at the scheduled time points. The concentration of NMD in rat plasma and tissues was determined by high-performance liquid chromatography, and the main pharmacokinetic parameters were calculated and compared. NMD was rapidly absorbed and reached the maximum plasma concentration in approximately 5 min after io administration. The absolute bioavailability after io administration was higher than that after ig administration (40.05% vs. 5.67%). There were significant differences in the tissue distribution of NMD with different administration routes. After io administration, NMD was distributed more in the lung, spleen, and brain tissues, and less in the kidney. The maximum drug concentration after io administration in the heart, liver, spleen, lung, kidney, and brain was 1.00, 0.47, 2.02, 1.47, 0.22, and 5.79 times higher than that after via ig administration, and the area under the curve value was 0.59, 0.78, 1.71, 1.84, 0.25, and 4.59 times greater, respectively. Nimodipine appears to achieve systemic effects via io administration. Compared with ig, io administration could significantly increase NMD distribution in the brain tissue, indicating that NMD could be delivered to the brain via io administration.

Comparative analysis of pharmacokinetics of vancomycin hydrochloride in rabbits after ocular, intragastric, and intravenous administration by LC-MS/MS.

The objective of this study was to compare the pharmacokinetics of vancomycin hydrochloride administered into rabbits through different routes and explore the feasibility of peptide drugs entering the systemic circulation through ocular administration. A convenient, accurate, and rapid liquid chromatography-trandem mass spectrometric (LC-MS/MS) method was established and used for the determination of vancomycin hydrochloride in rabbit plasma after intravenous administration (1.5 mg/kg), intragastric, and ocular administration (15 mg/kg). The pharmacokinetic parameters were analyzed using the DAS 2.0 software. We obtained a linear calibration curves vancomycin hydrochloride in plasma of rabbits over a concentration range of 0.05-10.0 µg/mL (R 2 > 0.9995), the interassay accuracy was within 5%, precision of 1.66-3.38%, and recovery of >85%. No matrix effects were observed. The absolute bioavailability of vancomycin hydrochloride after intragastric and ocular administration was 1.0 and 7.3%, with the half-life values of 63.1 and 138.5 min, respectively. Therefore, the LC-MS/MS method established in this experiment was suitable for the determination of vancomycin hydrochloride. Vancomycin hydrochloride was rapidly absorbed into the blood circulation after ocular administration. Ocular administration was linked to higher bioavailability compared with intragastric administration, suggesting that the former will become a route for the delivery of peptide drugs.

Systematic identification of chicken type I, II and III interferon-stimulated genes.

Interferon-stimulated genes (ISGs) play an important role in antiviral innate immune responses. Although many ISGs have been identified in mammals, researchers commonly recognize that many more ISGs are yet to be discovered. Current information is still very limited particularly for the systematic identification of type III ISGs. Similarly, current research on ISGs in birds is still in its infancy. The aim of this study was to systematically identify chicken type I (IFN-α), II (IFN-γ) and III (IFN-λ) ISGs and analyze their respective response elements. RNA sequencing (RNA-Seq) was employed to identify those genes with up-regulated expression following chicken IFN-α, IFN-γ and IFN-λ treatment. Two hundred and five type I ISGs, 299 type II ISGs, and 421 type III ISGs were identified in the chicken. We further searched for IFN-stimulated response elements (ISRE) and gamma-activated sequences (GAS) elements in the promoters region of ISGs. The GAS elements were common in the promoter of type II ISGs and were even detected in type I and III ISGs. However, ISRE were not commonly found in the promoters of chicken ISGs. Furthermore, we demonstrated that ISRE in chicken cells were significantly activated by IFN-α or IFN-λ treatment, and expectedly, that GAS elements were also significantly activated by IFN-γ treatment. Interestingly, we also found that GAS elements were significantly activated by IFN-λ. Our study provides a systematic library of ISGs in the chicken together with preliminary information about the transcriptional regulation of the identified ISGs.

Systematic Identification of Host Immune Key Factors Influencing Viral Infection in PBL of ALV-J Infected SPF Chicken.

Although research related to avian leukosis virus subgroup J (ALV-J) has lasted for more than a century, the systematic identification of host immune key factors against ALV-J infection has not been reported. In this study, we establish an infection model in which four-week-old SPF chickens are infected with ALV-J strain CHN06, after which the host immune response is detected. We found that the expression of two antiviral interferon-stimulated genes (ISGs) (Mx1 and IFIT5) were increased in ALV-J infected peripheral blood lymphocytes (PBL). A significant CD8+ T cell response induced by ALV-J appeared as early as seven days post-infection (DPI), and humoral immunity starting from 21 DPI differed greatly in the time scale of induction level. Meanwhile, the ALV-J viremia was significantly decreased before antibody production at 14 DPI, and eliminated at 21 DPI under a very low antibody level. The up-regulated CD8+ T cell in the thymus (14DPI) and PBL (7 DPI and 21 DPI) was detected, indicating that the thymus may provide the output of CD8+ T cell to PBL, which was related to virus clearance. Besides, up-regulated chemokine CXCLi1 at 7 DPI in PBL was observed, which may be related to the migration of the CD8+ T cell from the thymus to PBL. More importantly, the CD8 high+ T cell response of the CD8αß phenotype may produce granzyme K, NK lysin, or IFN-γ for clearing viruses. These findings provide novel insights and direction for developing effective ALV-J vaccines.

A novel flunarizine hydrochloride-loaded organogel for intraocular drug delivery in situ: Design, physicochemical characteristics and inspection.

We developed a safe and efficacious drug delivery system for treatment of brain diseases. A novel in-situ gel system was prepared using soybean oil, stearic acid and N-methyl-2-pyrrolidinone (NMP) (10:1:3, v/w/v). This system had low viscosity as a sol in vitro and turned into a solid or semi-solid gel in situ after administration. The poorly water-soluble drug flunarizine hydrochloride (FNZ) was incorporated into this "organogel" system. Organogel-FNZ was characterized by light microscopy, differential scanning calorimetry (DSC) and rheology. Drug release in vitro was investigated. The initial "burst" effect did not occur in organogel-FNZ, which is different from other gels formed in situ. Pharmacokinetic studies were undertaken in rats using gel administration (14 mg kg-1), intravenous administration (5 mg kg-1) and administration using drops (14 mg kg-1). Organogel-FNZ could reduce the clearance rate and prolong the duration of action, in the plasma and brain tissues of rats. The peak serum concentration, area under the curve and absolute bioavailability of the organogel-FNZ group were higher than those of the intraocular- drops group. Organogel-FNZ is a promising drug-delivery system for treatment of brain diseases by intraocular administration.

Drug delivery for intracerebral of flunarizine hydrochloride after intraocular administration in rats. / ç›é ¸æ°Ÿæ¡‚åˆ©å—ªç»çœ¼éƒ¨ç»™è¯åŽåœ¨å¤§é¼ è„‘å† çš„é€’é€.

OBJECTIVES: Intraocular administration was a commonly route of administration in clinic, but most of the intraocular administration was only used to treat local ocular diseases. Based on the particularity of the ocular structure, this article mainly explored the feasibility of flunarizine hydrochloride in the treatment of ischemic cerebral vascular diseases (ICVD) by intraocular administration. METHODS: A total of 150 SD rats were randomly divided into 3 groups with intraocular, intragastric, and intravenous administration, respectively. The doses were 14 mg/kg for intraocular and intragastric groups and 5 mg/kg for intravenous group. The plasma and brain concentration of flunarizine hydrochloride were analyzed by high performance liquid chromatography (HPLC). Main pharmacokinetic parameters and absolute bioavailability were evaluated. Brain targeting of flunarizine hydrochloride through intraocular administration was studied by drug targeting index of brain (DTIbrain). RESULTS: Maximum contentration (Cmax) and area under the time-concentration curve from o to t (AUC0-t) of plasma after intraocular administration were significantly higher than those of plasma after intragastric administration (both P<0.05). Cmax and AUC0-t of brain after intraocular administration were significantly higher than those of brain after intragastric administration (both P<0.05). The bioavailability of plasma and brain after intraocular administration was 18.67% and 34.67%, respectively, which was higher than 14.32% and 21.56% of plasma and brain after intragastric administration. The DTIbrain of intraocular administration was 1.84, and the DTIbrain of intragastric administration was 1.48. CONCLUSIONS: Flunarizine hydrochloride could be absorbed into the systemic circulation after intraocular administration. Not only the absolute bioavailability but also the brain targeting index of intraocular administration is higher than that of intragastric administration.

Effect of different doses of borneol on the pharmacokinetics of vinpocetine in rat plasma and brain after intraocular administration.

The effect of different doses of borneol on the pharmacokinetics of vinpocetine after intraocular administration in the rat plasma and the brain was investigated.Intraocular administration of vinpocetine (3 mg/kg) was performed, in combination with different doses (0, 5, 10, and 20 mg/kg) of borneol. Intravenous administration of vinpocetine was used as a control (1 mg/kg). The concentrations of vinpocetine in the rat plasma and the brain were determined using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Using the non-compartmental models with the DSA 2.0 software, the main pharmacokinetics parameters and the brain-targeting effect evaluated.In comparison with intravenous administration, after intraocular administration of vinpocetine alone, the absolute bioavailability (F) of vinpocetine was 43.82% for the plasma, and the drug target index (DTI) was 1.05 for the brain. After intraocular administration of vinpocetine combined with different doses of borneol, the relative bioavailability (Fr) of vinpocetine in the plasma was increased by 130.46-182.90%. The relative bioavailability (Fr) of vinpocetine in the brain was improved (147.19-225.36%). The DTI was 1.12, 1.18, and 1.21 for 5, 10, and 20 mg/kg of borneol, respectively.Compared with the intraocular administration of vinpocetine alone, the co-administration of different doses of borneol resulted in an obvious brain targeting effect.