Cancer metastasis often leads to death and therapeutic resistance. This process involves the participation of a variety of cell components, especially cellular and intercellular communications in the tumor microenvironment (TME). Using genetic sequencing technology to comprehensively characterize the tumor and TME is therefore key to understanding metastasis and therapeutic resistance. The use of spatial transcriptome sequencing enables the localization of gene expressions and cell activities in tissue sections. By examining the localization change as well as gene expression of these cells, it is possible to characterize the progress of tumor metastasis and TME formation. With improvements of this technology, spatial transcriptome sequencing technology has been extended from local regions to whole tissues, and from single sequencing technology to multimodal analysis combined with a variety of datasets. This has enabled the detection of every single cell in tissue slides, with high resolution, to provide more accurate predictive information for tumor treatments. In this review, we summarize the results of recent studies dealing with new multimodal methods and spatial transcriptome sequencing methods in tumors to illustrate recent developments in the imaging resolution of micro-tissues. The early stage of cancer is usually considered curable using surgical resection and therapeutic agents . TreatmeThe next-generation sequencing (NGS) has the advantages of large-scale and high-throughput gene sequencing  Figure . ResearcHowever, NGS has been largely directed toward sequencing malignant genes, which has mainly ignored important information of non-malignant samples involved in cancer cell progression in the TME . MoreoveThe subsequent use of single-cell RNA sequencing (scRNA-seq) has largely compensated for the aforementioned deficiency . NGS maiDue to the inability to detect whole tissue sections, important information between cells and tissues may be omitted . HoweverIn this review, we summarize studies on the use of spatial transcriptome technologies to characterize tumor metastasis and the TME, and describe future multimodal analysis methods that combine multiomics and multidimensional methods in spatial transcriptome technology.The use of scRNA-seq, first used in 2009, successfully detected multiple transcriptional variants expressed in the same cell [Just as the initial DNA sequencing method of Sanger and Gilbert led to genetic sequencing ,20, scRNFor tumor cells with high intratumoral heterogeneities (ITHs) , single However, a tumor is not independent of the rest of the body. Like other tissues and organs, it depends on the overall regulation system of the body during its formation. For example, the abnormal function of the extracellular matrix in the TME causes cancerous epithelial cells to lose polarity and adhesion, change morphology, and move to surrounding tissues .in vitro dissociation, therefore, scRNA-seq studies are limited to independent cell populations [While scRNA-seq separates the whole tissue sample into individual cells by analyzing a cell suspension, it loses the whole system of interactions with the body after ulations . For comIn addition, scRNA-seq itself has some technical limitations, including that freezing and dissociation experimental steps may lead to partial cell death . The varin situ hybridization (ISH) [in situ hybridization (FISH) using fluorescent probes to detect chromosome translocations [In 1969, Pardue and Gall developed on (ISH) , using nocations   is able to perform targeted detection of short-stranded RNA fragments in tissues with cells, to characterize the effects of cell–cell interactions in tissues on a single cell [The better the detection of genetic material in a single cell, the higher the localized imaging resolution of a single cell. The emergence of seqFISH has improved sequencing efficiency and cell imaging resolution. Next, Smart-seq2 continues to optimize detection of the single cell transcriptome, focusing on full-length coverage of transcripts, to improve cDNA production and expand establishment of a cDNA library  Figure . Except gle cell .The above methods are all directed to the detection of mRNAs. Although long-chain noncoding RNAs (LncRNAs) in cells are not involved in protein coding, they also play an important role in regulating transcription and the activity of proteins . Lv et aThe fluorescence microscope has been used to image cells in tissue sections, involving laser capture microdissection (LCM) , digitalin situ hybridization (MERFISH) technology, which could improve sequencing efficiency and minimize errors at the same time, to expand imaging of nearly 1000 different RNAs in hundreds of single cells, to achieve simultaneous improvement of both the efficiency and accuracy [in situ sequencing of transcriptome information of a single cell, as well as to amplify the target signal [Since then, SRM-based imaging technologies have been used in numerous studies. Chen et al. developed multiple error robust fluorescence accuracy . Wang ett signal . For thit signal  and usedin situ spatial transcriptome sequencing. The finer the classification of molecular probes, the more RNAs that can be detected. The higher the imaging resolution of these detection methods of tissue sections, the less the error between detection information and imaging, to achieve a higher degree of accuracy.Improvements of molecular probes and imaging technologies are the main factors in improving the accuracy and efficiency of in situ detection of transcriptomes within a single cell, investigators are no longer limited to studies of a single isolated cell, but can identify the influence between cells and the tissue environment, to evaluate the process of the transcriptome of the inner cells more comprehensively.In addition, with the improvement of the number and accuracy of in situ sequencing methods is often targeted at selected tissue sections, and it is difficult to detect whole tissues, especially those tissue areas that are difficult to target [However, the detection range of o target    method, in situ FISH of a whole tissue section. All mRNA information in the tissue is collected on a slide at one time. According to the cDNA collected in the spot on the slide, it is possible to determine what transcriptional information exists in the corresponding tissue section location, and to a large extent, the transcriptional information, cell positions, cell types, and the interaction within all tissues can be determined together.ST is therefore a spatial version of https://pages.10xgenomics.com/rs/446-PBO-704/images/10x_BR060_Inside_Visium_Spatial_Technology.pdf) [Although ST has improved the integrity of transcriptional information of tissue sections, resolution of a single cell was also reduced . This deogy.pdf)  are wideWe have summarized some applications of spatial transcriptome sequencing methods in tumors . It was In addition, more studies of ST of the TME have shown that tumor-related stroma cells in the TME are mainly cancer-associated fibroblasts (CAFs). However, myeloid cells, which are mainly tumor-associated macrophages (TAMs), also shared the same locations with tumors. It was found that M2 macrophages, CAFs, and some T lymphocytes were always located in the center of the tumor, and these T lymphocytes were identified as exhausted CD8+T cells  and uncoMetastatic tumor cells are therefore often accompanied by changes of cells in the TME, so there is a close relationship between tumors and the TME. A study of melanomas and the ST has also shown an interface between tumor cells and cells within the TME . These rin situ spatial sequencing. In contrast, ST with a large-scale sequencing advantage has reduced accuracy of single cell resolution. Hence, how to improve the single cell resolution of ST is the next goal of high-throughput spatial sequencing technologies. There are many ways to improve the resolution. One method involves continuously reducing the diameter of the spot from the abovementioned sequencing methods, and another is to improve the clarity of the imaging technology. Deconvolution is a mathematical algorithm that eliminates previous filtering, and reflects the original clarity of the image using imaging technology. Many imaging technologies therefore design the deconvolution algorithm to achieve high-definition imaging and restore the original image in tissue slices as much as possible.High resolution, but lack of tissue localization, are the characteristics of in situ sequencing of some key areas in the whole tissue section.We have summarized some research methods used to improve ST imaging during the past 2 years, basically involving deconvolution software designed to evaluate the localization of transcriptome expression in ST data through calculations, including SpaGCN , MULTILAThese techniques first detect the transcriptional information in cells, and then determine cell types using other information. Most cell types can be determined using this approach. However, some cell subtypes may share the same transcriptional information with other subtypes, but they are in different functional states.For example, the previously mentioned CAFs, which are normal fibroblasts in the overactivated TME, are constantly inducing remodeling of the structure and function of the extracellular matrix, followed by promotion of tumor metastasis . For CAFIn situ spatial RNA sequencing has already shown higher resolution of a single cell than does ST, so when combined with scRNA-seq, it may facilitate characterization of rare cell types. For example, by combining the data of smFISH and scRNA-seq, Massalha et al. [a et al. , in cholHowever, this approach is still limited to specific parts of tissues. We focus next on methods of combining scRNA-seq and ST, to directly map the data of cell types in whole tissue sections.The third way to improve resolution is to combine scRNA-seq with ST. The objective of scRNA-seq is to identify cell types, which also provides high resolution of single cells. The combination of these two methods could directly identify cell phenotypes in tissue sections, rather than first detecting transcriptional information in cells, then identifying the cell types; after identifying the cell type, we should be able to locate them in the tissue.In addition, using scRNA-seq, it may be possible to identify different transcriptional information in the same cell type, for example, distinguishing initial tumor stem cells, progeny tumor cells, and metastatic tumor cells, and then determining the different stages of tumor progression. It may be possible to identify similar transcripts in different cell types, for example, the tumor cells could transform to free mesenchymal cells during the EMT. It may also be possible to show that a cell may interact with adjacent cells to produce mixed transcriptional information; for example, tumor cells may promote their own development by encoding specific metabolites that can induce abnormal functions of stromal and immune cells .Like the difference between NGS and scRNA-seq, ST using NGS can be used to identify and locate the main cell types in tissue sections, but it cannot identify the specific subtypes without using further technology. The combination of ST and scRNA-seq can therefore ensure the high resolution of single cells and show their spatial localizations  and 3.We will review the development of integrating methods. Several deconvolution methods have been used for bulk RNA-seq data to analyze the signals of cell types . SubsequHowever, these deconvolution methods were based on pre-selected cell marker genes, which were common characteristics of most cell types, but which might omit some rare specific gene expressions, differential gene expressions between individuals, and random gene expressions of the inner cells , so analin situ spatial RNA sequencing [Investigators have developed deconvolution methods by using datasets of scRNA-seq to replace marker gene screening. For example, Wang et al. developed multidisciplinary single cell deconvolution, using scRNA-seq datasets instead of marker gene screening to evaluate cell types and proportions in bulk RNA-seq . Butler quencing , by analquencing . Giladi quencing    used CelOther methods are based on Bayesian theory, which is a mathematical framework that models reasoning and decision-making under uncertain conditions . These pAmong them, CARD developed by Ying et al.  is an inCompared with the above methods, which mainly depend on a large cell population to select high matching degrees of cell types, Missarova et al. developed geneBasis to filter-out the generally expressed genes, and then identified cell types with high specificity and sensitivity. The method mainly focuses on the characterization of rare cell types .In addition to the above methods, which involve expansions of scRNA seq datasets and optimization of integrating algorithms, to improve the imaging resolution, Liu et al. proposed a two-factor smoothing technique involving ST. This method improved the previous one-factor smoothing technique, added spatial sequencing as another factor, so it also used spatial and pattern combined smoothing, which aimed to decrease the noise and dropout events caused by multiple path sequencing  Table 3Table 3.In general, the integration of ST and scRNA-seq datasets to improve resolution of single cells in tissue sections is the main direction of development of spatial transcriptome sequencing methods.As the integrating methods of scRNA-seq data on ST become more accurate, investigators are no longer limited in using transcriptomics datasets, but can integrate the multimodal datasets into spatial sequencing, such as genomics, epigenetics, chromatin omics, and proteomics. Thus, more comprehensive cell information in tissues can be displayed using spatial sequencing technology.These multimodal data methods include the following. Biancalani et al. developed Tangram , which iin situ spatial transcriptomics sequencing. For example, in the early years, Eng et al. developed sequential fluorescence in situ hybridization, which was able to identify cell subtypes and receptor ligand pairs between adjacent cells by detecting multimodal signals of chromatin and gene expressions [Except for the combination of ST, there are many methods integrating multimodal data with ressions . Vu et aressions . Park etressions  (Table 4in situ transcriptomic sequencing or ST, or the combination of the two with other datasets, the imaging algorithms are similar, which aim to restore the original spatial location of different cell types, tissue morphologies, and structures in tissue sections as accurately as possible. Although there are few studies using multimodal data analysis methods, they can result in original information of tumor tissues, to provide a reliable basis for the implementation of effective therapies.It can be seen that whether it is In general, sequencing methods have experienced an unprecedented era of rapid development, from sequencing of gene fragments to sequencing of all genetic materials in the whole cell and even whole tissues. Current sequencing technology has been extended to an interdisciplinary analysis model that integrates multiple omics, multiple data, and multiple dimensions.This has provided many advantages. It greatly improves all cellular and non-cellular information in tissues, and visually displays as much microscopic information as possible. Using a tissue slide, it is possible to identify a certain cell type from multiple perspectives, and collect relevant information about this type of cell. The resolution of cells has been improved from multiple perspectives, and many rare cell phenotypes that were missed by single sequencing technology were found. This expanded cell datasets and increased the number of targets that can be used for new therapies.However, there are still limitations. Due to the IHT of tumors, more unknown tumor phenotypes may appear in the future, whereas current multimodal datasets are based on the statistics of known experimental research structures. Therefore, while studying new methods, we need to constantly update the information of clinical tumor specimens to keep up with changes of tumor evolution.The main purpose of spatial sequencing is to visualize the original data in tissue slices. Although multimodal spatial sequencing methods can analyze the information of tissue slides from various angles, these data also depend on tissue samples. Without tissue slides as the main body, these datasets merely comprise data that cannot be used. For tumor metastasis and the complex TME, this is a dynamic process. A tissue slide can only analyze limited information, but cannot track and record a dynamic process. In addition, a mouse model can reflect the evolution of tumors but may differ from human tumors.The production of sequencing technology ultimately provides information for the transition to clinical medicine. So how do we combine experimental methods with clinical applications? By identifying changes of cell types in tissue sections, we can predict tumor stages and the therapeutic effect for patients. Then, tissue samples at different stages can be selected before and after treatment, and spatial sequencing could be conducted to identify changes of tumor cells and cell types in the TME. It is also necessary to conduct long-term and continuous studies on the same patient or a class of patients with similar diseases. Previous studies have also used single sequencing techniques such as NGS and scRNA-seq to evaluate the therapeutic effect and resistance of patients ,116.However, these studies can only predict the treatment effect involving changes of certain gene expressions and cell types, and these predictions only reflect the data from the heat map, but spatial sequencing technology can directly map the data into tissue samples, which is equivalent to indicating the expression information of the tumor related genome, transcriptome, chromatin, protein, and DNA methylation in tissue samples at one time, so that we can intuitively observe these microscopic molecular changes when looking at the patient’s pathological sections. Thus, the accuracy of evaluation is improved to a certain extent. However, this method is more suitable for tissues and organs with obvious solid tumors. For tumor cells with systemic metastasis through the blood, SCS is still best for liquid biopsies .Overall, the use of spatial sequencing technology will promote the development of a personalized and accurate targeted treatment system for patients, to improve the survival of patients with tumor metastasis.


Hospital service area (HSA) and Hospital referral region (HRR) are significant in organizing maternal care resources in hierarchical medical systems. This quantitative study aims to delineate HAS and HRR by using obstetrics medical record data reflecting patients’ medical behavior to improve the efficiency of the utilization of medical resources. The Dartmouth method and an improved version that considers the administrative division was applied to delineate HSA and HRR by using the obstetrics medical records in Hubei Province of China in 2016. The result shows that 117 Dartmouth HSAs have a strong correlation with the county boundaries and 22 Dartmouth HRRs are highly coincident with the prefecture boundaries in Hubei. In addition, 25 improved Dartmouth HRRs within prefecture boundaries and core areas serving patients across prefecture boundaries have been identified. Based on the above results, two sets of hierarchical healthcare systems were constructed, respectively, which can provide methods and references for delineating HAS and HRR in the hierarchical medical systems in other regions of China and developing countries. The findings of this study shed light on future research and policymaking in the spatial organization of medical resources for improving the efficiency and equity in maternal care delivery. Improving maternal health is one of the Millennium Development Goals issued by the United Nations. The health of pregnant women in China is also an important issue in academic research and policymaking, especially since the implementation of the two-child policy in 2016 influenced the population of newborns. The births of second or later children, accounted for 45% of the newborns in 2016 [The medical systems in most countries are hierarchical, and many countries have formed medical referral systems . For exaIn China, the medical system is divided into three levels . PrimaryTherefore, an effective and reasonable hierarchical healthcare system is of great significance. Rational and reliable hospital service areas (HSAs) and Hospital Referral Regions (HRRs) are fundamental for the construction of a hierarchical healthcare system . HSA refThe Dartmouth method, which was created by the Dartmouth Institute for Health Policy and Clinical Practice, is frequently applied to divide HSA and HRR  and playMost studies focused on European and American countries, with few studies focusing on the construction of HSA/HRR in China. In addition, the above methods share a common feature, that is, in order to improve the degree of localized medical treatment, the HSA/HRR is different from the administrative planning boundary . This isIn recent years, the Chinese government has announced a series of policies and aims to build a medical association that includes hospitals of different levels and is actively attempting to create a hierarchical medical system on this basis. However, the division of medical associations is mainly based on empirical experience and does not consider patients’ personal preferences when choosing hospitals. High-quality, high-level hospitals or specialized hospitals with outstanding professional capabilities are always identified as the headquarters of medical associations . MoreoveIt can be seen from the above information that most existing HSA and HRR research are conducted in developed countries. The scale and structural characteristics of the population, the distribution of medical resources, the ownership of hospitals, and the planning and allocation of medical services in developed countries are different from those in developing countries . The patBased on the above research purposes, this study takes the maternal service in Hubei Province as an example. First, the classic Dartmouth method is used to divide HSA and HRR based on the registration data of pregnant women. The HRR is jointly composed of HSAs, and the minimum service population size in each HRR conforms to the actual situation of the study area, rather than directly applying existing empirical indicators. Therefore, HSA and HRR form a two-tier referral system. In addition, according to the characteristics of China’s resource allocation based on administrative divisions as the basic unit, this paper adds the conditions of administrative division into the Dartmouth method. Dividing intra-city HRRs according to cases seeking treatment inside the city they live, and cross-city HRRs based on women seeking treatment across the city. Besides, a cross-city HRR is formed by the merger of intra-city HRRs, these two types of HRRs also meet the same minimum service population size. HSA, intra-city HRR, and cross-city HRR form a three-tier referral system. Finally, this paper compares the similarities and differences between the two referral systems, which not only provides references for China’s medical planning but also provides references for HSA and HRR research in other developing countries.Hubei Province includes 17 cities in four regions: eastern, central, western, and northern Hubei . The datThe coordinates of 313 secondary hospitals and 108 tertiary hospitals were obtained based on their addresses using the geocoding Application Programming Interface (API) of Baidu Map. As shown in (1)Hospitals were assigned to a town in which they are located, and a town with any hospital was referred to as the hospital core.(2)Maternal flow data were spatialized, including the address of hospitals and residence sites, as well as the number of patients to hospitals.(3)Then each town was assigned to the hospital core which serves this town most.(4)All the towns assigned to the same hospital core constitute an original HSA(5)Spatial adjacency, administrative divisions and majority principles  were used to check and adjust the initial HSADartmouth method, Huff model, and network optimization method mainly aim at improving the degree of localized medical treatment ratio in the HSA\HRR. Therefore, the boundaries of HSA\HRR might be different from administrative boundaries, which is inconsistent with the Chinese policy of using the administrative divisions for healthcare resource allocation. In order to make HSA more in line with the actual situation in China, this study applies Dartmouth’s method considering the administrative divisions to identify HSAs:iL is the localized service index of the i-th HSA, k is the number of townships in the HSA, ijN is the number of patients in township j who choose localized medical treatment, and jN is the number of all the patients from township j.The meaning of the Localization index is the proportion of patients in the HSA/HRR who seek medical treatment locally . It can iL of each HRR is not less than 0.65 [There are three types of hospitals in China: primary hospitals, secondary hospitals, and tertiary hospitals. Among them, secondary hospitals and tertiary hospitals provide professional delivery services, with the medical resource quality of tertiary hospitals being the highest. So, in this study, secondary hospitals correspond to HSAs, and tertiary hospitals correspond to HRRs. The delineation of HRRs is similar to HSAs, however, HRRs need to meet the following conditions: The han 0.65 , and eacThe spatial and scale patterns are different between the secondary and tertiary medical flow . The totiL index varies from 0.50 to 0.98. According to Based on the Dartmouth method, there are 117 HSAs corresponding to secondary hospitals in Hubei Province , includiBased on the Dartmouth HRR method, a total of 22 HRRs have been delineated in Hubei Province . Each HRintra-city HRR;The above-mentioned HRRs based on all three levels of medical flows is different from the administrative unit. For example, the No. 13 HRR mainly served by Wuhan Southern Hospitals covers a large area, and for some parturients, travel cost is high. In order to construct HRRs that conform to the characteristics of China’s resource allocation, this study divides the three-level medical flow into the intra-city medical flow and cross-city medical flow, constructing the intra-city HRR  and the According to the tertiary medical flow inside the cities, Hubei province is divided into 25 intra-city HRRs. Each intra-city HRR contains an average of 4.3 hospitals, including five HRRs with one tertiary hospital, eight HRRs with two tertiary hospitals, four HRRs with three tertiary hospitals, and eight HRRs with four or more tertiary hospitals. The spatial distribution of intra-city HRRs is similar to the classic HRR, the number of intra-city HRRs in the eastern and central regions is greater but they are smaller than those in the western and northern regions. However, the layouts of the classic HRR and intra-city HRR also have differences. The intra-city HRRs have no super-large unit like No. 13 among the classic HRRs. In addition, the boundaries of intra-city HRR are more in accordance with city boundaries. Such a distribution pattern is convenient for health management departments to supervise and manage healthcare facilities.cross-city HRR;Based on the data of 14,830 parturients using maternal services across city boundaries, this study identified the pattern of cross-city HRRs, results are shown in In previous studies, most hierarchical medical systems had a two-tier structure, consisting of HSAs and HRRs. This study proposes to use HSA, intra-city HRR, and cross-city HRR based on the actual maternal data of Hubei, forming a three-tier maternal hierarchical medical system. However, there is only one cross-city HRR in Hubei Province, and its core region of maternal care is the intra-city HRR in south Wuhan . This meIn order to promote a more balanced development of quality maternal service, regional sub-centers were further identified in this study based on cross-city maternal data from tertiary hospitals. It was found that the intra-city HRRs codes 20 and 23, located in Jingzhou and Huangshi, are regional subcenters for parturient women who seek treatment across city boundaries. To a certain extent, the existence of these two sub-centers facilitates the medical treatment of puerpera in the west and north regions of Wuhan and relieve the pressure of obstetrics in southern Wuhan. However, there is still no regional sub-center providing maternal care services in northern and western Hubei.The two-tier maternal service referral system is composed of classic Dartmouth HRR and HSA. A total of 117 HSAs and 22 HRRs were identified in Hubei Province using the Dartmouth method, forming a total of 106 referral routes, that is, the patient can be referred to the corresponding HRR when utilizing healthcare services in HSAs within the HRR. The spatial distribution of the referral system and the referral relationship between HRR and HSA are shown in Considering the administrative divisions, the three-level referral system consists of HSAs, intra-city HRRs, and one cross-city HRR . That meThere are two main differences when comparing the two referral systems:The two types of referral systems have different numbers of referral routes. There are 106 referral routes from HSAs to HRRs and 113 routes from HSAs to intra-city HRRs. However, after taking cross-city HRR into consideration, referral routes are more diverse. Then the three-tier maternal service hierarchical medical system is refined. The classic HRR in the two-tier referral system crosses multiple city boundaries, and the situation is more complicated in terms of relocating medical resources. Additionally, it does not fully consider the western and northern regions with the tertiary obstetric medical resources in Hubei. The three-tier hierarchical medical system of maternal service promotes localized medical treatment and identifies the provincial center and sub-centers. This alleviates the high cost of referral travel in remote areas to a certain extent.This study finds that the Dartmouth method has certain applicability in China, which can identify the actual service area of different hospitals and referral systems according to medical flow data. However, attention should be paid when applying this method in the context of China. China is a developing country with higher population mobility and more frequent changes in planning. Moreover, China mostly takes the administrative unit as the basic unit for planning and resource allocation. The disparities in healthcare quality among various regions may be larger than those in developed countries. The reference value provided by Dartmouth HSA and HRR for medical resource planning could be limited.When Dartmouth HSA and Dartmouth HRR are used as a reference for future medical policymaking, it can ensure that all regions have a high proportion of localized medical treatment. However, HRR in developed regions only covers adjacent areas, which masks the reality that it also provides medical services for many patients in less developed regions who seek medical treatment across regions. Hospitals in cross-city HRR often need to serve both local patients and cross-city patients, which will be large in volume and may aggravate the differences in medical resources allocation among regions. Moreover, even if it is necessary, patients who choose hospitals from other cities, need to pay higher travel costs. Therefore, based on the two-tier referral system, the assessment, planning and optimization of medical resources will not provide the most reliable reference for policymaking. In addition, there is a big difference between HRR and the administrative division, so many original medical policies need to be adjusted accordingly. The professionals may also be needed to manage the medical resources in HRRs across prefectures and various HRRs’ medical resources within a prefecture.In order to achieve equitable development of medical resources among regions in the future, medical policies should refer to the existing three-tier referral system and reflect the actual medical utilization behavior of patients to a greater extent. Even if the challenge of a large number of cross-city medical treatments cannot be reduced in the short term, the establishment of regional medical sub-centers serving cross-city medical patients, can reduce the travel cost of patients to some extent and more efficiently allocate the medical resources among regions. For example, this paper identifies the regional sub-centers located in Jingzhou and Huangshi. If these two sub-centers are fully developed, patients in remote areas from Wuhan could seek treatment across-city in less time. However, how many such sub-centers need to be set up and how many medical resources need to be allocated in different regional centers need more in-depth research.Although the three-tier maternal service medical referral system is considered more comprehensive, this study still has some limitations: first, the division of HSA and HRR by the Dartmouth method is mainly analyzed from the perspective of the demand side. It does not consider how busy the hospitals are. Secondly, the cross-city medical treatment of parturients is affected by personal preferences and may not be necessary. Based on this division, the constructed cross-city HRR may not be the best fitting unit for tertiary hospitals. Thirdly, the tertiary obstetric resources in the southern area of Wuhan are abundant and high in quality. The different responsibilities of hospitals in this area cannot be determined in detail only through the division of HRR and HSA. Finally, the use of medical treatment flow data reflects the real medical treatment behavior and medical service utilization pattern, but the allocation of medical resources may be unequal and unreasonable in developing countries. In this context, the medical flow data may also have flaws in identifying medical service patterns.This study uses actual maternal flow data and comprehensively considers the relationship between administrative boundaries and HRR boundaries for delineating HSA and HRR in the hierarchical medical system. The Dartmouth method delineated 117 Dartmouth HSAs with strong correlation with the county boundaries and 22 Dartmouth HRRs highly coincident with the prefecture boundaries in Hubei. An improved version that considers the administrative division was applied to identify 25 improved Dartmouth HRRs within prefecture boundaries and core areas serving patients across prefecture boundaries for a three-tier medical service system. The first layer is the HSA corresponding to the obstetric department of a secondary hospital, which has a high coincidence rate with the county boundary. The second level is an intra-city HRR, which has a higher rate of coincidence with the city boundary. The third level is a cross-city HRR. If the three-tier medical referral system is used as the basic unit for future medical policymaking, it can not only reflect the service pattern of secondary hospitals and tertiary hospitals to the surrounding areas, but also further depict the spatial pattern of patients’ utilization of maternal care services across cities.These two sets of hierarchical healthcare systems were constructed, respectively, which can provide methods and references for other regions of China and developing countries. Although there are some limitations in this study, this research constructs HSA/HRR and a hierarchical medical system for China by combining the Dartmouth method and the characteristics of Chinese medical resources planning. It has enriched China’s research on the medical system and provided an important reference for the formulation of medical associations policies and hierarchical medical system policies.


Cerebral small vessel disease (CSVD) is one of the most important causes of vascular dementia. Immunosenescence and inflammatory response, with the involvement of the cerebrovascular system, constitute the basis of this disease. Immunosenescence identifies a condition of deterioration of the immune organs and consequent dysregulation of the immune response caused by cellular senescence, which exposes older adults to a greater vulnerability. A low-grade chronic inflammation status also accompanies it without overt infections, an “inflammaging” condition. The correlation between immunosenescence and inflammaging is fundamental in understanding the pathogenesis of age-related CSVD (ArCSVD). The production of inflammatory mediators caused by inflammaging promotes cellular senescence and the decrease of the adaptive immune response. Vice versa, the depletion of the adaptive immune mechanisms favours the stimulation of the innate immune system and the production of inflammatory mediators leading to inflammaging. Furthermore, endothelial dysfunction, chronic inflammation promoted by senescent innate immune cells, oxidative stress and impairment of microglia functions constitute, therefore, the framework within which small vessel disease develops: it is a concatenation of molecular events that promotes the decline of the central nervous system and cognitive functions slowly and progressively. Because the causative molecular mechanisms have not yet been fully elucidated, the road of scientific research is stretched in this direction, seeking to discover other aberrant processes and ensure therapeutic tools able to enhance the life expectancy of people affected by ArCSVD. Although the concept of CSVD is broader, this manuscript focuses on describing the neurobiological basis and immune system alterations behind cerebral aging. Furthermore, the purpose of our work is to detect patients with CSVD at an early stage, through the evaluation of precocious MRI changes and serum markers of inflammation, to treat untimely risk factors that influence the burden and the worsening of the cerebral disease. Deterioration of the central nervous system associated with age depends on different mechanisms related to chronic inflammation and the consequent decline in the immune response. With aging, the dysregulation of molecular mechanisms of the immune system promotes neurodegeneration and plays a crucial role in determining outcomes such as dementia and stroke. Cerebral small vessel disease (CSVD) is nowadays recognized as one of the most important causes of vascular dementia: immunosenescence and inflammatory response, with involvement of the brain’s vascular system, constitute the basis of this disease . The defAlthough the concept of CSVD is broader, this manuscript focuses on describing the neurobiological basis and immune system alterations behind cerebral aging. Furthermore, the purpose of our work is to detect patients with CSVD at an early stage, through the evaluation of precocious MRI changes and serum markers of inflammation, in order to treat untimely risk factors that influence the burden and the worsening of the cerebral disease.Aging is characterized by immune-related modifications that determine susceptibility to infectious diseases, increased risk of cancer and cardiovascular diseases, and reduction in the effectiveness of vaccines. These conditions depend on a decline in the immune system called “immunosenescence” .Immunosenescence identifies a condition of deterioration of the immune organs and consequent dysregulation of the immune response caused by cellular senescence, which exposes older adults to vulnerability .A low-grade chronic inflammation status also accompanies it without overt infections, a condition named “inflammaging”, inflammation is a valid mechanism to destroy micro-organisms and harmful substances entering the human body, but chronic subclinical inflammation status associated with senescence increases the risk of degenerative and metabolic disease .Under normal conditions, the innate and adaptive immune systems recognize dangerous substances and harmful stimuli such as pathogens   have adverse effects on tissue homeostasis by producing inflammation amplifiers. It seems that SCs promote inflammaging through a senescence-associated secretory phenotype (SASP), characterized by a broad series of inflammatory actors  and degradation products of extracellular matrix .The presence of cellular debris and nondegradable products in the tissues triggers the innate immune response through the molecular cascade of “damage-associated molecular patterns” (DAMPs) that recognize self-signals and produce macrophage activation .Innate immune activation promotes pro-inflammatory pathways through “nuclear factor kappa-light-chain enhancer of activated B cells” (NF-kB) and “signal transducer and activator of transcription” (STAT): the immune cells produce a significant number of inflammatory mediators (cytokines and chemokines) that amplify immune involvement .In the pathophysiology of ArCSVD, the state of chronic inflammation caused by cellular aging and dysregulation of the immune system leads to endothelial dysfunction and alteration of the blood–brain barrier: it seems that this process could be identified early through the detection of circulating biological markers  categoriMoreover, many studies have underlined that the early identification of pathological modifications of the BBB through imaging techniques such as classical MRI or functional MRI could predict the cognitive impairment in aged patients .BBB leakage and endothelial dysfunction are considered the progenitors in the development of ArCSVD. Early correction of the risk factors underlying these two alterations is considered the best treatment to halt cognitive decline.The BBB dysfunction leads to the release of the central nervous system (CNS) antigens into the peripheral circulation and the infiltration of leukocytes into brain tissue ,18.The transit of serum proteins at the neurovascular unit due to BBB dysfunction leads to microglia activation. Macrophages of microglia can produce chemokines, which provoke the migration of peripheral inflammatory cells to the CNS, generating a perpetual inflammatory microenvironment and supporting activated lymphocytes to meet CNS antigens ,22,23,24Therefore, immunosenescence and the set of altered molecular mechanisms described above are the basis for understanding many diseases in older people: cellular aging damages the immune system both in its innate and adaptive compartments.The innate immune response constitutes the first barrier of the human body against pathogens and harmful substances .The cellular elements of this compartment can recognize conserved pathogen-associated molecular patterns (nonself) and autologous antigens of the damage-associated molecular patterns (self) through different specific types of sensors .Beyond this, the revolutionary concept that has been increasingly understood in recent years is that the innate immune system has a kind of memory, named “innate immune memory”, which can recognize dangerous signals through a few receptors and amplify the response by using many effectors in a model known as “bow-tie” .Furthermore, it was observed that after some time from a specific activation , innate immune cells were able to recognize the trace of the previous activation and to react in the absence of BCG to other stimuli through this “innate memory” .The knowledge of these elements and complex mechanisms makes it possible to understand the complexity of the action of the innate immune system.The aging processes of innate immunity are related to dichotomous mechanisms: immune stimulation and immune paralysis, exacerbation of some functions coexisting with depression of others. This peculiarity, in the past, was considered the hinge of inflammaging through the excessive production of inflammatory mediators by senescent immune cells and the consequent boost of adaptive immunity and amplification of low chronic inflammation status. Recent observations, however, have shown that inflammaging is the result of many alterations such as mitochondrial aging, resident microbiota changes, and oxidative stress (oxy-inflammaging) .The trained innate immune memory described above could be, according to several studies, the promoter of immune activation in the absence of specific triggers. It is mainly constituted through epigenetic modifications and probably concerns the molecular pathways of energy production, thereby generating a significant change in the mechanisms of immunomodulation: macrophages, which are the protagonists of innate immunity, can modify their phenotype and the pattern of inflammatory molecules produced, playing a pivotal role in determining the state of low-grade chronic inflammation that characterizes the elderly, and it seems likely that many of these changes happen in the epigenomic field .Chronic stresses determine intracellular modifications and deterioration of molecular mechanisms over the years—mitochondrial dysfunction, impaired autophagy, and damage to DNA repair processes. It implies that, with aging, the balance between pro-inflammatory and anti-inflammatory molecules is lost, and there is a preponderance of inflammation status: the consequence of this mismatch is the damage of the whole organism at different levels . The counterbalance of chronic low-grade inflammation that represents the hyperactivation of the immune system in the elderly is immune paralysis. In physiological conditions, this mechanism protects against the activation of immune cells against autologous antigens and avoids self-damage. However, with aging, the functional alterations cause the loss of protective activity against pathogens and other harmful substances, reinforcing the condition of fragility that makes it more likely for an older adult to fall ill .However, the condition of immune paralysis provides some advantages in the elderly organism because it reduces the energy expenditure of the innate immune system: the most explicit example is macrophages, for which it is too expensive to maintain the M1 phenotype (pro-inflammatory and antitumor). At the same time, it is much more energetically convenient to acquire the M2 phenotype (angiogenetic and cancer-growth-promoting) .Therefore, the crucial task, for the aged organism, is to maintain a convenient balance between the need to fight pathogens and the pathway of chronic inflammation to avoid tissue damage. Mitochondria come into play in this challenging process. Some degree of mitochondrial dysfunction is responsible for successful aging as opposed to overall mitochondrial dysfunction, responsible for unsuccessful aging .Specifically, regarding the ArCSVD, the infiltration of immune cells such as macrophages, neutrophils, T cells, and NK cells causes inflammation, endothelial dysfunction and ischemia in the cerebral area .In addition, the infiltration of senescent immune cells worsens the inflammatory state around the ischemic region through increased expression of inflammatory markers and alteration of the immune system. Moreover, infiltrating immune cells can produce ROS (reactive oxygen species), which cause oxidative stress on endothelial cells, leading to vascular remodeling and impaired vascular tone .In the innate immune system, microglia is another important actor who plays an essential role in CNS phagocytosis, removing dead neurons, injured glial cells and debris of the myelin sheath. In aged people, the microglia also becomes senescent, and its dysfunction contributes to a low-grade inflammation state in CNS and neurodegenerative diseases ,35.Moreover, senescent microglia provokes impairment of phagocytosis and migration of immune cells, leading to senescent cells and debris accumulation. These cause chronic inflammation that damages cerebrovascular structures and neurons ,36.Ultimately, the alterations of the innate immune system responsible for the pathogenesis of ArCSVD are mainly due to the loss of the physiological synergy existing between immune and non-immune cells: chronic inflammation modifies the cellular microenvironment and triggers processes of cell degeneration that, together with the normal cellular aging, promote changes in the cerebral circulation.Endothelial dysfunction, chronic inflammation promoted by senescent innate immune cells, oxidative stress, and impairment of microglial functions constitute the framework within which small vessel disease develops: it is a concatenation of molecular events that promotes the decline of the central nervous system and cognitive functions slowly and progressively.The adaptive immune system also plays a crucial role in the immunological memory of the organism, which encourages the quick identification and elimination of specific pathogens by meeting specific sequences. With aging, two substantial changes occur: there is a reduction in naïve T cells resulting in a depletion of the TCR repertoire and an increase in memory T cells triggered by pathogens and aggressors previously met. In addition, naïve T-cell regeneration is limited because of thymic involution since puberty, continuous antigenic stimulations over the years, and reduced bone marrow supply for cellular production in the elderly  Figure .The above would explain the increased incidence of infections and cancer that characterizes aging and the decreased vaccination response .However, these concepts have been challenged over the years: it is not entirely true that there is a depletion of the TCR heritage with aging, and it has been found that the replenishment of naïve T cells is partly obtained by the action of interleukin-7 and by the contribution of the Stem-Cell-like Memory T . If, on Although the available data are conflicting, CMV-driven recurrent antigenic stimulation is responsible for successful aging, maintenance of adaptive immune system functions, and vaccination response .Concerning their features, aged T cells are divided into two classes: senescent and exhausted cells. Senescent cells are inert; exhausted cells are “dormant” and can be awakened by different stimulations by modulation of the surface receptors recovering their activity .This aspect is essential because of its clinical relevance. For example, there are some cancers of the elderly, which is possible a modulation through the action at the level of checkpoint inhibitors (the surface receptors described above) .Whatever stimulation activates the immune system, the molecular cascade of events that trigger the immune response is impaired in the elderly—alterations in immune synapse formation, mistakes in the process of constitution of transcription factors (mainly at the expense of those factors that are modulable). Among those described above, one of the most studied factors is a factor that regulates the assembly of molecules in cholesterol/ganglioside-containing nanoclusters .Therefore, changes considered as part of aging in the past are the expression of an environmental role in the human body modifications and suggest that lifestyle and nutrition affect the process of immunosenescence  Figure .Many of the changes determined by the immunosenescence depend on the changes that characterize the whole organism and the cerebral district with aging: in aged spontaneously hypertensive rats (SHR), we observed an increased expression of adhesion molecules at the level of the cerebral microcirculation, then increased recruitment of lymphocytes in this district and dysfunction of small vessels resulting in thrombosis .Moreover, antibodies against endothelial cells have been found to suggest the conditioning role of B lymphocytes in the genesis of endothelial dysfunction .In the interdependent relationship between immunosenescence and inflammaging, it has also been observed a pathway of monocyte/macrophage activation able to promote the production of reactive oxygen species (ROS), to reduce the production of nitric oxide (NO), thus causing the increased expression of adhesion molecules, the hypertrophy of wall smooth muscle cells and the remodeling of the extracellular matrix by metalloproteinases , chronicIn ArCSVD, old cerebral non-immune cells, such as endothelial cells, astrocytes, and oligodendrocytes, play an essential role in the destruction and dysfunction of the brain–blood barrier (BBB). Moreover, the immune cells can intercept these senescent cells and activate different responses. The endothelium is a critical system that plays a crucial role in the ArCSVD  through The dysregulation of the vascular tone is one of the most critical factors of endothelial dysfunction. It is mainly due to reduced production of endothelial nitric oxide synthase (eNOS)-derived NO ,52,53: mThe suppressed activity of eNOS causes modifications in vascular tone, reduced cerebral blood flow, increased oxidative stress and vulnerability to acute ischemia ,52,53.With aging of endothelial cells, therefore, a vicious circle is established through oxidative stress and inflammation, which progressively suppress the endothelial function and its ability to ensure a balance between vasodilating and vasoconstricting factors in favour of vasoconstriction, and this imbalance causes cerebrovascular dysfunction .Aging causes activation of the Toll-like receptor Nuclear Factor kappa-B (TLR-NF-kB) molecular pathway, leading to increased production of inflammatory cytokines such as IL-1a, IL-1b, IL-6, TNF-a, and IFN-γ .These inflammatory molecules and the NF-kB protein provoke the expression of nicotinamide adenine dinucleotide phosphate reduced (NADPH)-oxidase and, consequently, elevated levels of reactive oxygen species (ROS) .In addition, the reactive oxygen species induce positive feedback on NF-kB activity and can recall circulating and residential immune cells , causingVessel rarefaction is a common feature of ArCSVD, leading to a reduction in the tissue blood flow . The imaAnother factor involved in the pathogenesis of ArCSVD is dysfunction of the BBB due to the alterations of endothelial function and pericyte disorders. Immunosenescence induces those cellular changes described above, which, with other risk factors such as trauma or hypertension, provoke an increase in BBB leakage ,60.Although the pathophysiology of BBB dysfunction is complex, the most critical key factor of BBB leakage is endothelial dysfunction .The chronic exposition of endothelial cells to the shear stress leads to modifications of the tight junctions and increased permeability of BBB .As a result of BBB leakage, immune cells and circulating non-immune cells can infiltrate the brain tissue and provoke a low-grade inflammatory state . With agIn addition, the immune cells perpetuate the local inflammation and slow down the tissue reparation processes because of increased inflammatory cytokines and oxidative stress .Aging causes an altered molecular microenvironment, which leads to an altered immune response and modification of the immune system. However, few studies have examined the possible relationships between BBB dysfunction and age-related modifications of peripheral cells of the innate or adaptive immune systems. Furthermore, many reports have suggested a possible contribution of the BBB changes in neuroimmune dysfunction. Some works have suggested that the early identification of pathological modifications of the BBB in living humans through imaging techniques such as PET, SPECT, and MRI could predict clinical symptoms such as cognitive impairment in aged patients .Many studies suggest that modifications in BBB are one of the most critical factors contributing to the formation of white matter lesions and other secondary injuries in ArCSVD. These lesions found their radiological correspondents in the white matter hyperintensities (WMH) .Furthermore, there is a connection between WMH and BBB leakage: the more significant the range of WMH, the more severe the BBB leakage .In recent years, many studies have highlighted the role of Diffusion Tensor Imaging (DTI) MRI in CSVD ,67. DTI About the new frontiers of study in the field of the disease in question, in recent years, in vitro microfluidic BBB chips are becoming a kind of beacon and guidepost leading brain science research in the 21st century ,69,70.First of all, the multi-cell co-culture system provides potential for the investigation of CNS diseases. Because of the complex structure of the BBB, the conventional in vitro cell culture model fails to truly present the dynamic hemodynamics of BBB and the interaction between neurons. Therefore, miniaturized microfluidics-based BBB chips are commonly used to co-culture various cells on a small-sized chip to construct three-dimensional (3D) BBB or BBB-related organ disease models. By combining with other electrophysiological, biochemical sensors or equipment and imaging systems, it can, in real-time and quickly screen disease-related markers and evaluate drug efficacy .BBB chips are non-invasive in vitro models capable of early identification of BBB leakage, considered one of the main risk factors for developing ArCSVD.The detection of WMH on conventional MRI, the brain microstructural damage on DTI MRI and the presence of BBB leakage through in vitro BBB-Chips models could allow the early identification of patients who will develop ArCSVD, which is why early diagnosis and treatment of the underlying risk factors is crucial in blocking the development of the disease .Recent findings suggest that there is no one-way path in which immunosenescence produces inflammaging: indeed, there is a bidirectional pathway in which inflammaging induces and maintains immunosenescence and vice versa. This was understood by looking at what happens to T lymphocytes: in the elderly, there is an increase in memory of CD8+ T cells (in the past considered inert) characterized by the loss of naïve T cell surface markers, such as CD28 and CD27, and the appearance of new senescent markers such as KLRG1. Thus, the increase in the number of T memory cells, and consequently of B cells, with aging could be the expression of a chronic continuous antigenic stimulation analogous to the mechanism of inflammaging  .The wide diffusion of CMV implies that the organism uses its immune energies to limit this specific infection in life. Thus, the T cell heritage is filled mainly by CMV-memory T cells. Previously, they were considered inactive and inert, but challenging data suggest that they are functionally active and contribute to inflammaging .This chronic stimulation of the immune system causes the increase in the number of senescent T cells and the inflammageing; the consequence of chronic stimulation, however, is the appearance of exhausted cellular phenotypes with the remodeling of membrane receptors and the emergence of inhibitory receptors  .Moreover, the B cell population is compromised and unable to fight against new harmful pathogens , which is why there is a significant risk of developing infectious diseases and cancer .This situation represents the dog biting its tail: the production of inflammatory mediators caused by inflammaging promotes cellular senescence and the decrease in the adaptive immune response; vice versa, the depletion of the adaptive immune mechanisms favours the stimulation of the innate immune system and the production of inflammatory mediators leading to inflammaging.In this synoptic picture, it is possible to understand the physiopathological bases of the ArCSVD: inflammaging is recognized as an essential risk factor for vascular dementia and stroke by promoting the aging of the immune system and by acting synergistically with traditional risk factors  .The progressive vascular damage of ArCSVD patients produces the release of central nervous system antigens in the peripheral circulation, generating the recruitment of lymphocytes in the brain tissue and the consequent dysfunctions .In contrast, cerebral dysfunction is responsible for the progressive deterioration of the immune system, causing a continuous stimulation of the immune cells through the release of antigens and immunogenic molecules .Pro-inflammatory molecules and activated immune cells are involved in the atherosclerotic process caused by inflammaging . The correlation between immunosenescence and inflammaging is fundamental in understanding the pathogenesis of ArCSVD. With one-to-one correspondence, cellular aging and the loss of integrity of the blood–brain barrier favour the perpetual activation of the immune system, and the latter continuously worsens brain damage through a varied series of molecular alterations. Therefore, immunosenescence and inflammaging are two sides of the same coin and constitute a dynamic field of study and research to find therapeutic perspectives which interfere with these mechanisms ,77,78 F.At the brain level, it has been studied how the dysregulation of vascular homeostasis and the balance between the processes of vascular dilation and constriction, with the release of inflammatory cytokines, derive from endothelial dysfunction. These processes result from chronic inflammation, responsible at least partly for the onset of ischemic events. Endothelial dysfunction is a determinant key of vascular damage. Chronic inflammation induces the release of molecular mediators that trigger immunological reactions capable of stimulating and worsening brain damage, thus affecting the outcome of the disease .Endothelial dysfunction is an early marker of cardiovascular disease: many studies showed the role of endothelial dysfunction and arterial stiffness as surrogate markers of vascular health.Several works documented how, in diabetic patients suffering from microangiopathy, the indices of arterial stiffness (PWV and Aix) were higher than in the control group. In contrast, indices of endothelial function (RHI) and cognitive function (MMSE) were lower.The authors speculated a possible correlation between increased arterial stiffness and decreased endothelial function, and mild cognitive impairment in this population .Another scientific work, instead, evaluated the correlation between type 2 diabetes mellitus complicated by diabetic foot and the presence of white matter hyperintensity (WMH), also associated with alteration of omentin levels, and endothelial and cognitive performance indices ,81.In patients with diabetic foot, there was evidence of alteration of small vessels, as documented by the finding of hyperintensity of the white matter. Furthermore, the higher frequency of WMH lesions in these patients could be due to the higher degree of arterial stiffness, endothelial dysfunction and reduced serum levels of Omentin-1. Because of that, more robust cardiovascular prevention with angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, statins and aspirin, and the evaluation of the values of some adipokines could be necessary to identify the risk of brain-vessel dysfunction in the diabetic patient with organ complications .It is essential to identify therapeutic targets and try to interfere with the mechanisms of immunosenescence. These therapeutic strategies could be specific or nonspecific. Concerning nonspecific therapies, it would be necessary to modify the risk factors that act by promoting chronic inflammation with consequent endothelial damage. Therefore, cardiovascular risk factors could be controlled , evaluating the possibility of introducing anti-platelet and anticoagulant therapy according to the individual patient’s ischaemic and haemorrhagic risk .The use of specific categories of drugs could have a dual role, on the one hand, to allow the control of cardiovascular risk factors and, on the other hand, to limit inflammatory damage. Some studies have shown that therapy with statins, cilostazol, and ACE-i could stabilize endothelial cells and reduce the chronic inflammatory process .Additionally, in some clinical trials, statins have significantly reduced the risk of cerebrovascular events in patients with hypercholesterolemia . ACE-I-tShuzhen et al. proposed that lipoprotein-associated phospholipase A2 (Lp-PLA2) and superoxide dismutase (SOD) are independently related to cognitive dysfunction and injury. This means that the white matter hyperintensity (WMH) of ArCSVD could be considered a therapeutic target .Considering the role of chronic inflammation and immunosenescence in the pathogenesis of ArCSVD, it would be interesting to consider using drugs already used for other neuroinflammatory diseases such as multiple sclerosis  in order to interfere with the action of the cytokines and inflammatory mediators involved .About individual drugs, fingolimod is a drug commonly used in relapsing/remitting multiple sclerosis; it reduces circulating lymphocytes and, consequently, prevents them from escaping from the lymph nodes during a stroke. Therefore, this drug could contribute to the prevention of early infiltration of lymphocytes in the brain and could reduce thromboinflammation . Fingolimod was used during the acute phase of ischemic stroke, and it showed an improvement in microvascular permeability and secondary damage .Further studies are still needed to understand its precise role. In addition, a recent study found that fingolimod could induce the expression of VEGF in astrocytes. It, therefore, could stimulate the molecule S1PR3, which plays a role in the breakdown of the BBB and would be followed by the entry of pathogenic lymphocytes into the brain .Natalizumab is another drug used in multiple sclerosis, and it has the function of blocking α4-integrin, which regulates the presence of lymphocytes (mainly T cells) in the central nervous system. The ACTION study considered administering natalizumab within 9 h of the onset of the acute ischemic event symptoms, but it did not find the effects of natalizumab on infarct growth. However, patients who received natalizumab had excellent cognitive outcomes at 90 days, especially in the small ischemic lesion group [Dimethyl fumarate is also used in the treatment of multiple sclerosis. It has been documented that this drug is more effective and has fewer side effects than the other drugs tested. Above all, it has an essential role in the process of oxidative stress cells. This mechanism allows for the transcription of genes downstream of the activation of the antioxidant nuclear factor Nrf2 .Molecules that mediate pericyte-EC interactions, such as TGF-β, and platelet-derived growth factor-BB (PDGF-BB), have been proposed as targets for the treatment of neurological disorders. Pericytes play a vital role in regulating various microvascular functions, such as angiogenesis, preservation of BBB, capillary blood flow, and migration of immune cells to the brain .Some works have shown how pericytes can differentiate into neurons, microglia, and vascular cells after brain lesions in ischemic disease and hypoxia .A trial in mice investigated how implantation of pericytes in the brain increased cerebral blood flow and could reduce pathological deposition of Aβ122 .Thus, this evidence suggests that pericytes transplantation may be a promising approach for treating ArCSVD.Inflammation is increasingly recognized as a risk factor for dementia, stroke, and ArCSVD .As life expectancy continues to rise worldwide, the number of individuals living in the community with age-related diseases will increase, especially ArCSVD .Inflammation not only acts through the aging of the immune system, but it also promotes the main cerebrovascular risk factors  to trigger their damaging effects. For example, in ArCSVD patients, recurrent injuries such as mild stroke lead to BBB loss, central nervous system antigens release into the peripheral circulation, and infiltration of lymphocytes into brain tissue and related brain dysfunction. Brain dysfunction can further damage the immune system, forming a vicious cycle .For this reason, the study of the role of the immune response during aging in the development of small vessel disease and the corresponding brain damage is of fundamental importance.The role of immunosenescence in endothelial dysfunction and blood–brain barrier disorder has led to exciting aspects. Therefore, it could be a possible candidate for further study ,101,102.To fully understand the pathophysiological mechanisms and therefore to obtain effective therapeutic strategies against ArCSVD, it is necessary to have a holistic approach; it is necessary to counteract the known risk factors, and it is necessary to limit the effects of age-related alterations on the immune system. Moreover, through new therapeutic strategies, it is necessary to limit the effects of chronic age-related inflammation that represents an essential trigger for oxidative stress and endothelial dysfunction. The early diagnosis of ArCSVD, through serum markers of inflammation or functional or conventional MRI techniques, is crucial to treat early and strictly the risk factors that can cause disease progression and the onset of dementia.Because the causative molecular mechanisms are not yet fully elucidated, the road of scientific research is stretched in this direction to discover as many aberrant processes and to ensure therapeutic weapons can improve the quality of life of people affected by them.