Small intestinal neuroendocrine neoplasms (siNENs) are slowly growing tumours with a low malignant potential. However, more than half of the patients present with distant metastases (stage IV) and nearly all with locoregional lymph node (LN) metastases at the time of surgery. The value of locoregional treatment is discussed controversially.In stage I to III disease, locoregional surgery was currently shown to be curative prolonging survival. In stage IV disease, surgery may prolong survival in selected patients with the chance to cure locoregional disease besides radical/debulking liver surgery. It may improve the quality of life and may prevent severe local complications resulting in a state of chronic malnutrition and severe intestinal ischaemia or bowel obstruction.Locoregional tumour resection offers the opportunity to be curative or to focus therapeutically on liver metastasis, facilitating various other therapeutic modalities. Risks and benefits of the surgical intervention need to be balanced individually. Neuroendocrine cells, distributed throughout the body and also diffusely scattered in the gastrointestinal (GI) tract, may give rise to neuroendocrine neoplasms (NENs). As demonstrated in a prospective, 1-year incidence study, 44 out of 306 (14.4%) newly diagnosed gastro-entero-pancreatic NENs of the GI tract were located in the small intestine .•.1•].In some of these stage IV tumours, the type and location of the primary tumour may remain unknown even after extended, additionally performed cross-sectional imaging (e.g. MRI) or endoscopy of the upper and lower GI tract as the second and third steps of the investigation pathway.As shown recently , the sitLiver biopsy not only confirms the NEN itself and helps to identify the tumour site of origin but also facilitates tumour grading Table . Liver lThe majority of primary tumours are slowly growing submucosal lesions, only a few millimetres in size (60% ≤ 20 mm), which invade the lamina propria or submucosa or the muscularis propria and are classified as pT1 or pT2  [However, the latter endoscopic diagnostic modalities are not widely available. They may be effective but are time-consuming and expensive. Their role in routine staging before planning treatment must require clearer definition, whilst from the practical point of view, they seem to be of minor importance , 40.Very early in the course of the disease, mesenteric LNs metastases are invariably involved as single or multiple metastases, regardless of the size of the primary tumours, and are verified in up to 88% along the superior mesenteric vessels , 41. LN Extra-abdominal LN metastases were observed in 24 (4.0%) of all patients at diagnosis and 37 (6.1%) patients displayed extra-abdominal metastases (other than metastatic LNs) at diagnosis ••.Whilst primary tumours are rarely visualised on CT, mesenteric LN metastases typically and often appear as contrast-enhancing soft-tissue masses with fibrotic bands radiating outward into the mesenteric fat in a stellate pattern surrounding LN metastasis •.LN metastasis tends to develop a “desmoplastic stromal reaction”, which is the result of coordinated changes in several stromal cells under the control of a single gene product, the CD36 protein. The repression of CD36 leads to a decrease in fat accumulation and an increase in matrix deposition •.Obliterative processes with considerable variation in the degree and rate of progression of “desmoplasia” may cause vascular encasement. Clinically, this may lead to abdominal pain, disabling diarrhoea, weight loss to the extent of malnutrition and eventually the risk of life-threatening situations with acute intestinal obstruction or intestinal gangrene.The current TNM classification Table  subdividIn the current pathological LN classification stage, pN1 by definition includes those patients with less than 12 regional LNs affected without mesenteric mass(es) greater than 20 mm in size. Patients with 12 or more positive regional nodes and/or mesenteric mass(es) greater than 20 mm in maximum dimension are defined as pN2. The number of involved LNs is of prognostic relevance .The number of resected and histopathologically examined and involved LNs and the LN ratios  was prognostic for overall survival—patients with 12 or more resected and examined LNs had the best survival outcomes .A lower metastatic node ratio predicted improved survival on multivariable analysis and is associated with high-volume institutions which are more experienced in LN dissection •.The WHO classification is not helpful in clarifying the possibility of radical LN dissection, an important step in treatment decision-making. The neighbourhood of the LN bulk and possible vascular involvement are to be assessed by CT/magnetic resonance imaging (MRI) angiography .Ohrvall et al. •• proposIn their series ••, 24% oThese findings were confirmed by Lardière-Deguelte et al.  who retrA key issue in siNEN resection is not necessarily the primary tumour itself. Surgery is to focus on preserving bowel function whilst selectively resecting mesenteric LNs. LN metastases have to be removed by cautious dissection around the superior mesenteric vessels in order to preserve the vascular supply to the small intestine and thus to avoid overextensive bowel resections. LN dissection along the vessels may be technically challenging, especially in complex situations with level and LN involvement. An appropriate preoperative LN cartography with respect to the jejunal vascular collaterals along the superior mesenteric vessels could help to estimate the technical demand of surgery. Radical lymphadenectomy along the superior mesenteric vessels may not necessarily require extended small bowel resection, which may cause “short bowel syndrome” and compromise patients’ quality of life , 49••.Skip metastases (LN metastases outside the “expected” lymphatic drainage) may occur in up to two thirds of patients, which may mandate more extensive lymphadenectomy to prevent unresectable locoregional recurrence .Patients with local tumour-related symptoms generally undergo local resection at the time of diagnosis. However, some symptomatic patients have advanced level 4 LN metastases, encasing the superior mesenteric vessels and rendering radical resection challenging. In highly advanced situations including the infeasibility to remove the LN bulk with an acceptable level of morbidity, stenting and other palliative methods improve the clinical symptoms and prevent bowel ischaemia .The liver and the peritoneum are the two most common distant metastatic sites. With regard to the site of distant metastasis , 80/219 Solitary liver metastases are the exception. In only 1 of 40 patients, a solitary liver lesion with curative outcome after resection was documented .The majority of liver metastasis is multiple and often less than 10 mm in diameter. Because of their typical bilobar growth pattern, many resections of hepatic metastatic NENs that are considered curative at the time of surgery are palliative .At the time of initial diagnosis, fewer than five metastases in one lobe were revealed in 80 (21.9%) of 366 patients, 105 (28.7%) patients had bilobar and/or 5 to 10 metastases and 139 (38.0%) patients had more than 10 liver metastases, respectively. Ninety-nine (42%) patients without liver metastases at initial diagnosis developed liver metastases during follow-up [Morphologically, three different patterns of liver infiltration by metastasis are to be differentiated, since they have an impact on the therapeutic approach , 53•: inA bilobar pattern can still be approached surgically when 70 to 90% of the metastasis can be removed (cytoreduction/debulking) •, 54. HoBesides liver metastases in 48 (57.1%) of 84 patients, solely peritoneal and bone metastases were observed in 2 (2.4%) and 1 (1.2%) patients, respectively .Ovarian metastases occur in 4% of patients  followed by pancreatic 0.5%) and splenic metastases (0.5%) [% and splThe presence of carcinoid heart disease and mesenteric LN metastases, distant abdominal LN metastases, liver metastatic burden, extra-abdominal metastases, skeletal involvement and PC are independent prognostic factors for overall survival in siNENs ••. Bone Although occurring in only approximately 5%, lung metastases may impact patients’ outcome. The development of metachronous lung metastasis is associated with concomitant disease progression in established abdominal metastasis in most patients .The specific oncological characteristics of siNENs emphasise the necessity of exact (T)NM staging prior to planning further treatment. This is the prerequisite before selecting any treatment regime.In general, routine morphological radiological imaging techniques  tend to significantly underestimate the neuroendocrine disease .Preoperative morphological imaging “understaged” the disease in 14/20 (70%) when compared with intraoperative findings. In patients with multifocal primary tumours and miliary liver metastasis, no imaging modality was able to detect the entire extent of disease spread .The currently preferred hybrid imaging method combines the advantage of CT or MR and SSA receptor imaging in demonstrating the anatomical/morphological details of a lesion and its functional characteristics. The further development of radioactively labelled derivatives of synthetic SSA and the improvement of positron emission tomography (PET) technology, with greater resolution and simultaneous specific binding of radiopharmaceuticals to various somatostatin (SS) receptor subtypes, have improved the staging of siNENs compared to SS receptor scintigraphy alone.68Ga-DOTA-SSA PET/CT or MRT is recommended for functioning imaging in G1 and G2 tumours. This technique facilitates the diagnosis and extent of LN and distant metastases, such as peritoneal, liver and bone lesions [ lesions .Whole-body scans enable more exact staging and also the diagnosis of primaries in up to 89% of cases .18FDG-PET/CT is better suited for G3 tumours, which generally have a higher level of glucose metabolism and less SS receptor expression than low-grade NENs, and additionally provides prognostic information.However, single “hot spots” in functional imaging do not exclude multicentric disease. Therefore, if surgery is indicated, meticulous intraoperative abdominal examination with bidigital exploration of the small bowel, the liver and the abdominal cavity is to be performed as palpation is superior to any imaging technology .Differences in preoperative hybrid imaging and intraoperative findings may influence the surgical strategy.Positive LNs and distant metastasis in preoperative imaging do not per se exclude patients from surgery if they are fit for such treatment (heart disease not present or treated efficiently), and the siNEN itself or the distant metastasis is graded as G1/G2 ••.Traditionally, the approach of choice is laparotomy with meticulous exploration of the entire abdominal cavity excluding or verifying small extrahepatic metastasis with bidigital small bowel palpation to exclude multifocality and to determine the exact anatomy of LN metastasis to the superior mesenteric vessels .Laparoscopic procedures with the intention of curative surgery remain controversial •, 41, 62In palliative intent, the laparoscopic approach seems to be advantageous for the resection of local disease in patients with unresectable LN and liver metastases .Although primary siNENs may have an indolent clinical local course for a long time, radical locoregional surgery should be discussed in all patients with locoregional siNENs, with or without distant metastasis, who fulfil the basic criteria •, 63, 64The aim of locoregional prophylactic surgery in M0 and M1 patients is to improve quality of life by averting abdominal complications caused by unpredictable progressive local tumour growth which may ultimately result in bowel obstruction with the consequence of emergency surgery. Growing LN metastasis with fibrosis around the main vessels may result in a state of chronic malnutrition and severe intestinal ischaemia. These life-threatening local complications can be avoided by LN dissection as long as only LN levels 1 to 3 are affected , 65••.Depending on the location and number of the primaries, the optimal surgical treatment consists of sparing segmental small bowel or ileocaecal resection  with dissection of the regional LNs along the superior mesenteric vessels up to the inferior border of the pancreas (stage = levels 1 to 3 ).In patients with stage I to III disease, resection of the primary tumour and regional lymphadenectomy is indicated as a curative procedure which improves survival •.In patients with radically removed positive LNs (pN1M0), the 1-, 5- and 10-year survival rates were 100%, 100% and 83 ± 15%, respectively .The completeness of LN dissection also influences prognosis. Both overall survival and relative survival were significantly improved in the patients whose metastatic mesenteric LNs were grossly radically removed (R0) as compared to those with remaining pathological mesenteric LNs (R1) ••.After curative surgery, there is no indication for specific medical treatment, and there is no proven role for neoadjuvant or adjuvant medical treatment in si-NEN patients.Surgery is recommended in selected patients in the “simple (type 1) pattern” of liver involvement with the chance to cure locoregional disease alongside synchronously performed radical liver surgery.In stage IV disease with complex (type 2 or 3) liver involvement or unresectable extrahepatic distant metastasis, locoregional surgery is indicated in locally symptomatic patients •.Generally not considered to be curative, the value of locoregional surgery in asymptomatic patients is discussed controversially. After propensity score matching patients with and without prophylactic locoregional resection, Daskalakis et al. found no survival benefit comparing the two groups ••.The first systematic review of the literature described a clear trend towards improved survival after resection ••. AdditThe results of all the reviews and meta-analyses must be interpreted with caution due to potential selection and publication bias. The data support the consideration of surgery, particularly in patients with low tumour load and good functional status.Because large, randomised prospective studies investigating the management of primary tumour and regional LN metastasis in association with liver metastases are lacking, the optimal treatment strategy remains debatable and should be discussed individually in multidisciplinary tumour boards.Resection of liver metastasis may improve survival , 72, 73 Factors influencing prognosis are unclear. Manguso et al. • recentlThe 5‐year rate of overall survival was 79.4% for NR, 84.7% for PR and 100% for CR, demonstrating a trend that CR was the best, followed by PR then NR. However, 10‐year overall survival showed no significant differences . More than 10 liver lesions or receiving chemotherapy were negative predictors of survival.The minimal criteria required for liver surgery with “curative intent” are resectable G1/G2 liver disease with acceptable levels of morbidity (< 20%) and mortality (< 5%), the absence of right heart insufficiency and of extra-abdominal metastases (previously assessed by hybrid imaging), the absence of diffuse PC and a locoregionally resectable or already resected tumour.Locoregional resection performed by an experienced surgical team following a standardised surgical technique with low morbidity and mortality  has the With regard to peritoneal metastases, surgical resection is recommended synchronously with the primary tumour, LN and hepatic metastasis, if feasible, to control symptoms •, 64.In stage I to III disease, locoregional surgery may be curative. In stage IV disease, surgery is recommended in selected patients with the chance to cure locoregional disease besides radical liver surgery in the “simple (type 1) pattern” of liver involvement. In stage IV disease with complex (type 2 or 3) liver involvement or unresectable extrahepatic distant metastasis, locoregional surgery is indicated in locally symptomatic patients. Resection of the primary tumour should also be attempted in locally asymptomatic patients as the overall outcome may be better in patients after primary tumour resection even with distant metastasis although a direct causal relationship has not been proven to date. The type of surgery should be individualised, and no general approach can be recommended. The risks and benefits of the surgical intervention and the merits of pharmacological treatment (e.g. SSAs) have to be balanced. As such, the treatment of such patients is driven primarily by surgeons’ experience, and available data are based predominantly on retrospective studies. Keeping this in mind, current recommendations advocate for patients with siNENs  to be managed by multidisciplinary teams in experienced centres.


Recent technological advances have enabled spatially resolved measurements of expression profiles for hundreds to thousands of genes in fixed tissues at single-cell resolution. However, scalable computational analysis methods able to take into consideration the inherent 3D spatial organization of cell types and nonuniform cellular densities within tissues are still lacking. To address this, we developed MERINGUE, a computational framework based on spatial autocorrelation and cross-correlation analysis to identify genes with spatially heterogeneous expression patterns, infer putative cell–cell communication, and perform spatially informed cell clustering in 2D and 3D in a density-agnostic manner using spatially resolved transcriptomic data. We applied MERINGUE to a variety of spatially resolved transcriptomic data sets including multiplexed error-robust fluorescence in situ hybridization (MERFISH), spatial transcriptomics, Slide-seq, and aligned in situ hybridization (ISH) data. We anticipate that such statistical analysis of spatially resolved transcriptomic data will facilitate our understanding of the interplay between cell state and spatial organization in tissue development and disease. Characterization of the spatial context of cells and their cellular states is essential to understanding the connection between tissue organization and function, particularly in complex organs such as the mammalian brain. Furthermore, spatial context plays an important role in development and organ formation in multicellular organisms, as well as in aberrant processes such as cancer . Likewise, spatial variation in cellular density could also potentially mask spatial variation in gene expression . It is, however, important to identify variations in gene expression magnitudes across cells that do not arise from variations in cellular density. Alternative approaches such as SpaOTsc can accommodate nonuniform cellular densities if provided with geodesic distances . MERINGUE encodes these adjacency relationships using a binary adjacency weight matrix W with a weight of 1 if two data sets are adjacent and 0 otherwise  across the population of N cells (x) between spatially adjacent cells (Wij = 1) are positively correlated, Moran's I will be positive  for each gene (x) in a given cell (i) is positively correlated with that cell's spatially adjacent neighbors, the cell's LISA for the given gene will be highly positive. Again, LISA has a closed form, allowing P-values to be derived quickly. As such, MERINGUE defines the percent of cells with statistically significant LISAs as the percent of cells driving a spatially heterogeneous gene expression pattern. This use of LISA guards against the identification of spatially heterogeneous genes driven by small hotspots or outliers. Simulations suggest that false positives may be effectively eliminated by restricting to spatial heterogeneity driven by >5% of cells .To further characterize the scale of significant spatial gene expression heterogeneity, using the same matrix of normalized gene expression magnitudes and adjacency weight matrix x) in a given cell (i) is positively correlated with another gene's expression magnitude (y) in the cell's spatially adjacent neighbors (j), the SCI for this gene pair will be positive. MERINGUE computes this spatial cross-correlation index for all gene pairs to derive a spatial cross-correlation matrix that is then used for hierarchical clustering and dynamic tree cutting to group these genes into primary spatial patterns  in a given cell (i) of cell type A is positively correlated with the corresponding ligand gene's expression magnitude (y) in cells of cell type B among the cell's spatially adjacent neighbors (j), the inter-cell-type SCI for this cell type pair will be highly positive. Statistical significance can then be assessed by permutation testing . Indeed, when we applied MERINGUE to analyze 7365 genes among 260 spots, of the 834 identified as significantly spatially heterogeneous genes  driven by >5% of spots , 90% (754/834) overlapped with genes that are significantly differentially expressed genes across cell layers  as identified from ANOVA testing. Furthermore, these 834 spatially heterogeneous genes can be further partitioned into five primary spatial patterns that correspond to cell layers and combinations of cell layers as expected . One well-characterized aspect of spatial organization in the MOB involves the convergence of axonal projections from olfactory receptor neurons expressing a given olfactory receptor (Olfr) into glomerular neuropils at fixed locations in the glomerular cell layer of the olfactory bulb , rendering assessment of spatial heterogeneity for individual Olfr genes infeasible, by aggregating the expression of all detected Olfr genes, we validate that MERINGUE was able to identify significant spatial heterogeneity . The spatial expression pattern further corresponded approximately to the glomerular and surrounding cell layer as expected . For Slide-seq data of the mouse cerebellum, we applied MERINGUE to analyze 9762 genes among 1589 beads previously annotated to correspond to the Purkinje layer . We validate that Aldoc  is identified as among the most significantly spatially heterogeneous genes  , consistent with observations from the original publications  data of the mouse main olfactory bulb (MOB) and Slide-seq data of the mouse cerebellum . We found the resulting significance of spatial heterogeneity in terms of −log10 to be highly correlated across genes between all tested computational methods  . The resulting set of significantly spatially heterogeneous genes identified by each tested computational method using a common significance threshold  were also highly overlapping . We further evaluated the computational efficiency of each method in terms of runtime and memory usage as a function of the number of genes and the number of cells in the data set . We found that MERINGUE achieves improved computational efficiency compared to previously published computational methods . Thus, MERINGUE is capable of identifying spatially heterogeneous genes consistent with previously published approaches in a scalable manner.We next compared MERINGUE to previously published computational methods for analyzing spatially resolved transcriptomic data, SpatialDE and SPARK . Owing to its use of a distance-agnostic binary weight matrix, MERINGUE's resulting significance of spatial heterogeneity across genes was highly correlated between the uniform and nonuniform case as expected (Spearman's ρ = 0.862) . Likewise, although MERINGUE was able to identify 834 significantly spatially heterogeneous genes  in the uniform density case, 544 (65%) of these genes were recovered in the nonuniform density case with the same adjusted P-value and spot percentage thresholds. The discrepancies between the uniform and nonuniform cases can be largely attributed to changes in the binary weight matrix . Because SpatialDE and SPARK incorporate Euclidean distances between cells in their evaluation of spatial patterns, we reasoned that spatial variations in cellular density would impact their ability to identify spatially heterogeneous genes. We thus applied the same uniform and artificially induced nonuniform case comparison. As expected, the resulting significance of spatial heterogeneity across genes was less well correlated between the uniform and nonuniform density case for both SpatialDE (Spearman's ρ = 0.427) and SPARK (Spearman's ρ = 0.418) . Likewise, although SpatialDE was able to identify 360 significantly  spatially heterogeneous genes in the uniform density case, only 56 (16%) of these genes were recovered in the nonuniform case with the same adjusted P-value threshold. Similarly, SPARK was able to identify 664 significantly  spatially heterogeneous genes in the uniform case, but only 66 (10%) of these genes were recovered in the nonuniform case with the same adjusted P-value threshold.We developed MERINGUE to accommodate the nonuniform cellular densities inherent to tissues. Thus, we reasoned that changes in cellular densities should not substantially impact MERINGUE's ability to identify spatially heterogeneous genes. To assess MERINGUE's robustness to spatial variations in cellular densities, we artificially induced nonuniformity in the spatial distribution of ST spots by distorting their positional coordinates (x–y) plane, z-direction information can be obtained through optically scanning through imaging planes or sequential tissue sections. To show integration of z-direction information, we first applied MERINGUE to 3D in situ hybridization (ISH) data, aligned across multiple stage 6 Drosophila melanogaster embryos for 84 selected marker genes  as expected. We further validated that these genes could be grouped by MERINGUE into 14 primary spatial patterns that correspond to known regionally confined developmental fates and layers of the segmentation gene network , whereas pattern 13 corresponds to the thoracic segments and includes known gap gene Kruppel (Kr) (even skipped (eve) and odd skipped (odd), respectively  in at least one section out of 6214 genes tested . Because the distance between cells across serial sections are greater than the distances between cells within sections, we sought to identify spatial patterns consistent across layers by testing for spatial correlation between mutual nearest neighbors in space across sections . Such a multilayer integrated approach confirmed 242 significantly spatially heterogeneous genes as being consistent across sections . Of the remaining 172 genes that were identified as significantly spatially heterogeneous in individual sections but not across sections, visual inspection showed that although these genes show spatial variability within sections, there was minimal correspondence across sections . Such transcriptional patterns may be indicative of layer-specific subpopulations or transcriptional features. For structurally stereotypic tissues, consistency across tissue sections may be used as an additional criterion for identifying functionally relevant spatial patterns. Thus, MERINGUE is capable of accommodating 3D information to identify spatially heterogeneous genes in 3D as well as genes with spatial expression patterns consistent across serial sections.Alternatively, z-information may be derived through serial sections. We thus next applied MERINGUE to spatial transcriptomic data of four consecutive histological sections of a human breast cancer biopsy , we were able to identify at least one such spatially heterogeneous gene in 34 of 83 cell types and subtypes analyzed . None of the blank control barcodes, DAPI, or poly(dT) signals were identified as consistently spatially variable. MERINGUE further identified significant spatial gene expression heterogeneity within neuronal subtypes in both the anterior and posterior of the preoptic region. Likewise, spatial gene expression heterogeneity was identified in both inhibitory and excitatory neuronal subtypes. These aspects of spatial heterogeneity were consistent with previous published spatially unaware variance and principal components-based analyses and visual inspection  signals as negative controls within each cell type and subtype to identify additional aspects of spatial heterogeneity. Applying a rigorous approach to identify genes with spatially heterogeneous expression patterns that are consistent across tissue layers and reproducible across animals  . Esr1 and Irs4 have been previously shown to display sex-differences in their expression , but tachykinin receptor 1  was identified as significantly spatially heterogeneous in excitatory subtype E-8 neurons only in male and not female mice . Likewise, we confirmed that the fraction of cells expressing Tacr1 in E-8 neurons is not significantly different between male and female mice . However, when we quantified the fraction of cells driving the spatial heterogeneity of Tacr1 expression based on LISA, we observe a significant difference between male and female mice . Tacr1 knockout mice have been previously observed to show deficits in sexual behavior  between two neighborhoods  in the adjacency representation W (Methods): (1/dij + 1) + 1. Again, use of such a neighborhood representation can accommodate the nonhomogenous density of cells in tissues compared to a Euclidean distance-based measure of spatial distance. In this manner, if two cells are closer in space , their transcriptional similarity will give greater weight in the graph-based clustering. Incorporating these spatial weights into our graph-based clustering with all other parameters kept constant, we were able to split the cluster of cells expressing either eve or odd into two subpopulations, as desired . In a biological setting, however, cells from the same cell type may populate spatially distinct locations, but such distinct spatial locations alone would not necessarily indicate the presence of finer subtypes. We thus suggest that such spatially informed clustering to be complementary to differential expression analysis, whereby identified spatially distinct cell subpopulations should be analyzed for significantly differentially expressed genes to ensure the presence of significant, likely subtle, transcriptional differences. Therefore, by incorporating spatial information, in conjunction with differential expression analysis, we can identify finer, transcriptionally and spatially distinct subpopulations.Briefly, as in expression-based clustering, we constructed a neighbor graph in which nodes are cells and nodes are connected with an edge if the represented cells that are within the Supplemental Methods). We found that among the eight most populous inhibitory neuronal subtypes (clusters with >100 cells each), our spatially informed clustering was able to produce comparable clusters with the exception of I-2 and I-11, which were each split into two subtypes , consistent with our observations of significant spatial heterogeneity in Nos1 expression among I-11 neurons. Indeed, the two I-11 subtypes appear to be spatially distinct with C10 positioned more medially and C5 more laterally in the posterior preoptic region . Previously, I-2 neurons were observed to overlap with both the sexually dimorphic nucleus of the preoptic area (SDN-POA) as well as other anatomical nuclei such as the bed nucleus of the stria terminalis (BNST) . I-2 neurons were previously observed to show sexually dimorphic activation during mating and aggression based on the expression of immediate early gene Fos . Therefore, by incorporating spatial information via graph weighting, MERINGUE provides an alternative approach to tease apart spatially distinct subpopulations without impacting other transcriptionally distinct subtypes.Having shown that incorporation of spatial information via graph weighting can be applied to identify finer transcriptionally and spatially distinct subpopulations of cells, we next sought to apply this approach to identify finer neuronal subtypes in the preoptic region profiled by MERFISH . However, unlike the spatial autocorrelation function, this inter-cell-type spatial cross-correlation function is not solvable; thus significance must be assessed using permutation to derive a null model. We enhance computational efficiency by implementing a parallelized, adaptive permutation testing approach and assess significance using a permutation-based random label null model. We confirm using simulations that such a permutation-based assessment produces the expected type-I error rate .Spatially resolved transcriptomic data offers opportunity to identify gene expression patterns that may be indicative of putative cell–cell communication between spatially colocalized cell types. Previous computational approaches for inferring cell–cell communication from single-cell RNA-sequencing data have relied on correlations or coexpression of receptor genes in one cell type and corresponding expression of ligand genes in another cell type  in beads corresponding to Purkinje layer and expression of its ligand Ptn (secreted growth factor pleiotrophin) in spatially adjacent beads , suggestive that the Ptprz1 expression may be attributed to Purkinje cells within the Purkinje layer beads. Likewise, we confirm significant coexpression of Ptn with Slc1a3 , a glutamate transporter expressed by Bergmann glia . In contrast, restricting to well-detected ligand genes in the Purkinje layer beads and well-detected receptor genes in the spatially adjacent beads  enriched inhibitory I-2 neurons displayed substantial spatial overlap with estrogen receptor (Esr1) enriched neuronal subtypes. CYP19A1 is an enzyme that converts testosterone to estrogen, thereby modulating steroid hormone signaling in the preoptic region. The spatial organization of these CYP19A1-enriched neuronal subtypes with ESR1-enriched cells suggest that estrogen synthesized by these CYP19A1-expressing neurons may be interacting with estrogen receptors on spatially adjacent ESR1-expressing cells in a paracrine manner. To quantitatively assess to putative cell–cell communication between neuronal subtypes and spatially neighboring cells via such paracrine signaling, we applied MERINGUE to assess for significant spatial cross-correlation between Cyp19a1 expression in all neuronal subtypes and Esr1 expression in spatially adjacent neurons. Indeed, we are able to identify statistically significant spatial cross-correlation between Cyp19a1 expression in I-2 neurons and Esr1 expression in spatially adjacent neurons in a manner that is consistent across tissue layers and reproducible across animals . Furthermore, we also apply MERINGUE to test for spatial cross-correlation between Cyp19a1 expression in all neuronal subtypes and androgen receptor (Ar) expression in spatially adjacent neurons and do not identify consistently significant associations , thereby highlighting the nonrandomness of the Esr1 juxtaposition. This thus highlights MERINGUE's potential to quantitatively and systematically identify complementary gene expression patterns that may be indicative of cell–cell communication.We next sought to identify gene expression patterns that may be indicative of putative cell–cell communication between cell types using single-cell resolution MERFISH data of the preoptic region. Previously, Tacr1 expression in excitatory E-8 neurons and identified additional neuronal subpopulations within inhibitory I-2 and I-11 neurons with spatially distinct organization that may play roles in murine sexual behavior. MERINGUE is highly scalable and computationally efficient compared to previous spatial analysis methods . Furthermore, MERINGUE is robust to spatial variations in cellular density and can thus better accommodate nonuniform cellular densities common in tissues.Spatially resolved transcriptomic measurements demand computational approaches to identify and characterize significant spatial gene expression heterogeneity. Here, we presented MERINGUE as a density-agnostic approach to characterize spatially heterogeneous gene expression by identifying genes with spatially autocorrelated expression and gene pairs with spatially cross-correlated expression. We validated our approach by analyzing spatially resolved transcriptomic data from both sequencing and imaging-based methods in 2D and 3D to recover known biologically relevant spatial patterns. Our analysis of the mouse preoptic region by MERFISH revealed sexually dimorphic spatial organization of In comparison with previously published spatial gene expression analysis methods, although MERINGUE identifies and groups spatially heterogeneous genes into primary spatial patterns, it does not interpret identified spatial patterns based on predefined aggregated or alternating spatial patterns. In this manner, we find MERINGUE to be complementary to previously published spatial gene expression analysis methods in characterizing the spatial patterns of spatially heterogeneous genes. Likewise, we find MERINGUE to be complementary to expression-based clustering analysis to identify additional aspects of spatial heterogeneity within cell clusters or shared spatial gradients across cell clusters. In addition, in analyzing spatially resolved single-cell gene expression data sets obtained from different technologies, MERINGUE may also be applied in combination with different normalization and error model schemes such as cell volume-based normalization for imaging data  normalization without log transforming was applied to all spatially resolved transcriptomic data sets. For MERFISH data, RNA counts were normalized per cell by the imaged volume of each cell per the originally published analysis .If the data are produced by a mechanism that inherently induces some autocorrelation, such as high variability between spatially segregated batches or presence of noisy hotspots, then such a null hypothesis would not be appropriate, and evaluation of significance must be performed using permutation. We showed at least for a random subset of genes in our tested data sets that the null hypothesis is appropriate and thus results in essentially identical x identified as significantly spatially heterogeneous, MERINGUE next quantifies the scale of the spatial pattern by calculating the local indicators of spatial association . Downstream analyses can be restricted to spatially heterogeneous genes of a sufficient scale, defined by default as 5% of cells.For a given gene Again, these calculations were implemented in C++ using Rcpp  between all pairs of these genes. For We visualized these primary patterns by interpolating across spatial regions not covered by cells using Akima interpolation  will be given higher weight and cells farther apart  will be given a smaller weight: weight = (1/distance + α) + β, where α and β are pseudocounts to guard against excessively large and small weights, respectively. By default, we used α = β = 1, though the unit and magnitude of both α and β will depend on the unit and magnitude of distance. We then apply Louvain graph-based clustering to the resulting weighted graph  between expression of receptor x for the N cells of cell type A and the expression of ligand y for the M cells of cell type B:
Wictij = 1 if a cell of cell type A and a cell of cell type B are inferred to be adjacent or vice versa, and Wictij = 0 otherwise, to capture only spatial cross-correlation patterns between the two cell types.For each receptor-ligand pair, we computed an inter-cell-type spatial cross-correlation (iSCI with the likelihood of observing such an extremely positive value under a permutation-based random labeling model randomly permuting cell labels. To enhance computational efficiency, we allowed for parallelization across multiple cores and used an adaptive permutation testing approach whereby receptor-ligand pairs are first assessed for significance with 100 permutations by default, and putatively significant hits with permutation P-values < 1/100 are then reassessed with 1000 permutations and so forth. Additional gene pairs with known interacting products such as hormone-receptors can also be evaluated by this approach.We assessed statistical significance by comparing the observed An interactive application built on Shiny (


Thy1-YFP-16 reporter mice that express yellow fluorescent protein (YFP) in a broad spectrum of neurons. Swelling along damaged axons forms varicosities that fill with YFP. The frequency and size of axonal varicosities were significantly increased in the corpus callosum (CC) and cingulum at 3 days after the final blast exposure, versus in sham procedures. CC immunolabeling for reactive astrocyte and microglial markers was also significantly increased. NG2CreER;mTmG mice were given tamoxifen (TMX) on days 2 and 3 after the final blast to induce fluorescent labeling of newly synthesized myelin membranes, indicating plasticity and/or repair. Myelin synthesis was not altered in the CC over the intervening 4 or 8 weeks after repetitive blast exposure. These experiments show the advantages of transgenic reporter mice for analysis of white matter injury that detects subtle, diffuse axon damage and the dynamic nature of myelin sheaths. These results show that repetitive low-level blast exposures produce infrequent but significant axon damage along with neuroinflammation in white matter.The potential effects of blast exposure on the brain health of military personnel have raised concerns and led to increased surveillance of blast exposures. Neuroimaging studies have reported white matter abnormalities in brains of service members with a history of blast exposure. However, blast effects on white matter microstructure remain poorly understood. As a novel approach to screen for white matter effects, transgenic mice that express fluorescent reporters to sensitively detect axon damage and myelin remodeling were exposed to simulated repetitive blasts (once/day on 5 consecutive days). Axons were visualized using  TBI associated with high explosive blasts accounted for approximately one-third of the nearly 400,000 military TBIs between 2001 and 2008.3 The effects of repetitive exposure to occupational blasts are an ongoing concern within the military. The National Defense Authorization Act (2018–2020) called for occupational surveillance to evaluate blast exposure, for example, among those engaged in breaching exercises or firing of heavy artillery.4 Early studies indicated a potential correlation between military personnel exposed to repeated low-level blast exposure and worsened long-term neurological and cognitive outcomes.6Traumatic brain injury (TBI) is a significant public health concern for civilian and military populations. Brain injury associated with an explosive blast raises additional concerns due to the unknown effects of blast exposures and the potential for long-term symptoms.7–9 Studies of blast-exposed veterans with and without TBI found that blast exposure decreased DTI fractional anisotropy in several white matter tracts.10 Both breachers and veterans examined more than 1 year after blast exposure with TBI exhibited DTI changes in white matter.12 Further, veterans with blast-related mild TBI and loss of consciousness had increased white matter abnormalities and cognitive impairments.13The long-term effects of blast exposure on brain health remain poorly understood. Magnetic resonance imaging (MRI) studies using diffusion tensor imaging (DTI) detected changes in major white matter tracts of the brain in some deployed soldiers who experienced TBI with blast exposure.15 Tau, a protein that is bound to microtubules in the cytoskeleton of healthy axons, has been reported to become hyperphosphorylated and form aggregates in neurons in the brains of some veterans who experienced repetitive head injury and blast exposure.16 Additionally, brains from military service members with a history of blast exposure exhibited astroglial scarring at interfaces between tissues of differing densities; this astrogliosis involved the subpial glial plate, penetrating cortical blood vessels, structures lining the ventricles, and white matter–gray matter junctions.17 Together, these neuroimaging and neuropathology studies indicate vulnerability of the brain to blast exposure and the potential for axons and white matter to be critical pathological factors. However, the full range of neuropathological effects of repetitive blast exposure is challenging to determine because potentially subtle pathological features may be sparsely distributed throughout the brain, and may change over time. To overcome these challenges, we evaluated the effects of blast exposure on axon and white matter pathology using two lines of transgenic mice with genetic reporter constructs that enable detection of in vivo changes in specific cellular processes.Post-mortem studies of brains of service members who experienced blast-related TBI have identified multiple neuropathological features. Axon damage, identified as axon swellings with accumulated β-amyloid precursor protein (β-APP), has been observed along with disruptions to the myelin that ensheaths axons.Thy1-YFP-16 reporter mice express yellow fluorescent protein (YFP) in a broad spectrum of neurons so that individual axons can be visualized in diverse brain regions, including the corpus callosum (CC).18–21 We used Thy1-YFP-16 reporter mice to screen for individual damaged axons across the brain and brainstem. YFP-filled enlargements form at sites of damage and result in swelling and formation of varicosities along axons. The localization of YFP in axon swellings is a sensitive indicator of axonal injury ranging from small swellings observed early, when damage may still be reversible, through to intensely fluorescent terminal end bulbs of disconnected axons.23 Our previous work in Thy1-YFP-16 mice demonstrated YFP detection of traumatic axonal injury in white and gray matter regions of the brain at 3 days after single or repetitive concussive TBI.21 Importantly, in comparison with β-APP immunohistochemistry used for standard neuropathology, confocal analysis in Thy1-YFP-16 mice can visualize YFP in three dimensions to identify terminal end bulbs at sites where axon damage has progressed to disconnection.2425–29NG2CreER;mTmG transgenic fluorescent reporter mice enable in vivo analysis of newly formed myelin sheaths over a specific time interval.30 The NG2 promoter is expressed in oligodendrocyte progenitor cells (OPCs) that proliferate and differentiate into myelin-forming oligodendrocytes. Tamoxifen (TMX) administration activates Cre recombinase that switches membrane fluorescent labeling from red to green in OPCs and their progeny. TMX given to NG2CreER;mTmG mice on days 2 and 3 after TBI results in green fluorescent protein (GFP) labeling of newly generated oligodendrocytes and their myelin sheaths.21 We previously showed that a single concussive TBI increased formation of new myelin in the CC through 4 weeks, yet CC atrophy was evident when survival was extended to 8 weeks after TBI.21 In this study we leveraged the advantages of the NG2CreER;mTmG mice to examine new myelin formation over 4 and 8 weeks after repetitive low-level blast exposure.Larger axons in white matter are wrapped by myelin sheaths that increase the efficiency and speed of action potential conduction. Myelin can be damaged by direct insults or as a secondary effect of axon damage. New myelin can be generated during activity-dependent adult plasticity and can form after injury or disease through regeneration and replacement of lost myelin sheaths.Thy1-YFP-16 and NG2CreER;mTmG mice was tested using an Advanced Blast Simulator (ABS) to model the blast wave component of low-level exposures to explosives or heavy artillery relative to potential effects of repetitive blast exposures during deployment or training. Studies of service members have shown that experiencing three or more TBI events, with or without blast exposure, may increase effects compared with those with fewer or no TBI history.32 In mice, our prior studies of concussive mild repetitive TBI, with impacts once per day on each of 5 consecutive days, resulted in persistent neuroinflammation in the CC and adjacent cerebral cortex.20 Therefore, the current studies in transgenic reporter lines used repetitive low-level blast exposures given once per day on each of 5 consecutive days for analysis of axon damage, myelination, and neuroinflammation.The effect of blast waves on the brains of NG2CreER BAkik/J), mT/mGROSA -Gt(ROSA)26Sortm4Luo/J), and Thy1-YFP-16 16Jrs/J). Mouse strains were bred as in-house colonies to generate experimental mice. First-generation offspring from NG2CreER and mTmGROSA breeders were used as NG2CreER;mTmG mice. The total number of Thy1-YFP-16 mice used was 15  and the total number of NG2CreER;mTmG mice used was 14 . Male and female mice were combined for analyses after separate analyses showed no sex-based differences. Littermates of Thy1-YFP-16 mice or NG2CreER;mTmG mice were used for quantitative analyses. The number of mice for each condition in each experiment is provided in each figure legend.All mice were treated in accordance with guidelines of the Uniformed Services University of the Health Sciences (USUHS) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were socially housed as 2–4 mice per cage in 35 cm × 16.5 cm × 18 cm cages that contained enrichment objects and maintained a 12-h daytime light cycle (0600–1800) during which all experimental procedures were performed. The following mouse strains were obtained from the Jackson Laboratory : 34 The rSham mice were anesthetized and the same procedures were followed except that the blast was not deployed. There was no significant difference in mean peak incident overpressure , mean duration (7.25 ± 0.235 msec), or mean impulse (62.9 ± 0.795 psi-msec) across the five blast exposures for any groups. Righting reflex time was significantly prolonged in rBlast mice (mean = 78.55 sec) compared with rSham mice (mean = 45.8 sec)  = 206.6; p > 0.0001). Righting reflex time did not change significantly across the 5 days of rBlast or rSham procedures for either genotype or injury condition.At 8–9 weeks of age, mice were randomly assigned to repetitive sham (rSham) or repetitive blast (rBlast) procedures. The rBlast mice were exposed to five consecutive simulated blast exposures, at 24-h intervals, delivered in an ABS . ABS procedures were performed with mice in the prone position for frontal blast exposure. Each mouse was anesthetized with 3% isoflurane and the head and body were secured to a wooden tongue depressor, wrapped in gauze, placed in mesh to prevent rotational movement, and positioned to face the oncoming blast as previously detailed.Thy1-YFP-16 mice were perfused with 4% paraformaldehyde and brains were cut as 14-μm-thick cryosections.21 For qualitative analysis of the distribution of axon damage, sagittal sections of Thy1-YFP-16 mice were imaged using a Zeiss Axioscan Z.1 (Carl Zeiss) with a Plan-Apochromat 20 × objective to image the entire brain section, from olfactory bulb to brainstem. To examine the three-dimensional (3D) structure of YFP-filled axons, confocal image stacks were acquired on a 700 laser scanning confocal microscope (Carl Zeiss) with a Plan-Apochromat 40 × /1.4 oil or 63 × /1.4 oil objective and processed using Vision4D Modular Software .At 3 days following the final rBlast or rSham procedure, 36 Pilot analyses in rSham and rBlast mice confirmed that axon diameter varied along normal-appearing axons but ranged from 0.5 to 1.5 μm. Therefore, quantification of axon damage included only those YFP-filled swellings >2.5 μm in width with peak fluorescent value (measured as maximum pixel intensity) of at least 25% greater than adjacent axons, which was above the 95% confidence interval of the YFP fluorescence intensity of adjacent normal-appearing axons. Cell bodies were distinguished using 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Axonal varicosities included YFP-filled swellings along an axon and terminal end bulbs, that is, swellings at the end of an axon segment. Analysis included six to eight sections per mouse.Quantification of axonal varicosities focused on regions of interest (ROIs) in the CC and cingulum in coronal sections at levels between +0.5 and −0.5 mm relative to bregma. The CC ROI extended from the midline bilaterally to the turning point of ventral descent in the external capsule. The cingulum ROI was evident as fibers oriented perpendicular to the underlying CC and distinct from the superior cortical tissue. Images acquired on an Olympus IX-70 fluorescence microscope with a SPOT RT3 camera  were analyzed using Image J software . The ROI area was measured with a 10 × objective. Morphological measurements of YFP-filled swellings used images acquired with a 40 × objective. The width of each axonal varicosity was measured in the plane transverse to the length of the axon. The width of CC axons was <1.5 μm as measured by electron microscopy.21 Astrocytes were evaluated by immunostaining for glial fibrillary acidic protein . Microglia/macrophages were identified using polyclonal rabbit antibody against ionized calcium binding adaptor molecule 1. All tissue sections were counterstained with DAPI nuclear stain . For quantification, images within the CC ROI were acquired with a 10 × objective on an Olympus IX-70 microscope using a SPOT RT3 camera. ImageJ software was used to threshold fluorescence levels to quantify the area of immunolabeling above background.28 Images were also acquired with a 40 × objective to examine the morphology of IBA1 immunolabeled cells to compare the density of resting or activated cells.37 Analysis included six to eight sections per mouse.Mice perfused at 3 days following final rBlast or rSham procedures were further analyzed using immunohistochemistry in coronal sections at levels between +0.5 and −0.5 mm relative to bregma. The CC ROI extended from the midline bilaterally to under the peak of the cingulum.NG2CreER;mTmG littermates received rBlast or rSham procedures. TMX  was administered via oral gavage on day 2 and 3 after the final blast or sham procedure.38 TMX induces Cre recombinase mediated deletion of a stop codon switches transcription from constitutive expression of membrane-localized tdTomato (mT) to NG2 driven expression of membrane-localized GFP (mG). Fluorescence was quantified in coronal sections at levels between +0.5 and −0.5 mm relative to bregma. The CC ROI extended from the midline bilaterally to under the peak of the cingulum.21 Images were acquired on an Olympus IX-70 microscope using a SPOT RT3 camera. ImageJ software was used to threshold the signal to above background and quantify the area of membrane fluorescence from the CC ROI, as previously detailed for CC mG labeling and for immunolabeling of myelin.28 Confocal images were acquired with a 40 × objective to examine the morphology of membranes labeled with mG and mT. Additional tissue sections were immunostained for total myelin based on myelin oligodendrocyte glycoprotein .Cohorts of t test was used to compare immunolabeling of astrocytes and microglia between groups. Two-way ANOVA with Sidak's post hoc multiple comparisons test was used at each time-point for comparing mG fluorescence and CC width between rSham and rBlast in NG2CreER;mTmG mice. Statistical significance was set at values of p < 0.05.Mice were allocated to the rBlast or rSham procedures using the random number generator function in Microsoft Excel. Investigators were blinded to animal injury condition during tissue analysis until after completion of the data analysis. GraphPad Prism 8.0 software (RRID:SCR_002798) was used for statistical analysis and graphing. Bar graphs demonstrate means with standard error of the means (SEMs) as well as symbols for individual mouse values. Two-way ANOVA with repeated measures, for CC and cingulum within same subjects, and Sidak's post hoc multiple comparisons test were used for axonal varicosity comparisons. An unpaired Student's Thy1-YFP-16 fluorescent reporter mice that express YFP in diverse neuronal populations but in only a subset of those neurons, which allows visualization of individual axons , but appears to vary with the degree of head immobilization.43 Indeed, single ABS exposure at 15 psi using a mesh to position the mouse in the prone position and minimize head movement resulted in a low-level blast frontal exposure that did not induce detectable microglial activation in the CC.33 The current studies used this same mesh procedure to similarly secure and position the mouse in the ABS. Our prior studies of concussive mild TBI in mice demonstrated persistent neuroinflammation in the CC and adjacent cortex after one impact per day on 5 consecutive days.20 With repetitive exposure pattern and increasing the ABS exposure to approximately 20 psi, we find a significant but low-level neuroinflammatory response in both astrocytes and microglia (To model the blast wave component of low-level exposures to explosives or heavy artillery, the current experiments used an ABS device to generate controlled high-fidelity simulation of both positive and negative phases of “free-field” blast.icroglia . This CCThy1-YFP-16 mice facilitated detection of axonal varicosities, including terminal end bulbs, which are significantly increased at 3 days after the final ABS exposure in the CC and cingulum (46 Axon damage after blast exposure appears to be subtle and not readily detected by immunohistochemistry.47–49 The current YFP approach revealed individual damaged axons in mice even when exposed to a relatively low mean peak incident overpressure of approximately 20 psi (cingulum  and 2. Py 20 psi .50 Studies with open field explosive blasts reported CC pathology with increased silver staining of damaged axons and ultrastructural evidence of myelin disruption.40 However, this electron microscopic technique to evaluate myelin can only sample small regions. Additionally, the ability to monitor the dynamic turnover of myelin membranes is important to capture in evaluating potential myelin repair. In the current study, the use of NG2CreER;mTmG mice enabled quantification of in vivo myelin synthesis during specific intervals after blast exposures. During these periods, rBlast exposure did not produce late-phase effects on synthesis of new myelin membranes or induce CC atrophy (51Our findings of acute neuroinflammation and axon damage in the CC led us to further examine myelination as an indicator of longer-term white matter injury after rBlast that is consistent with neuroimaging DTI findings in patients who have been experienced blasts. Myelin has been shown to be disrupted in TBI and myelin repair may have profound implications for recovery of axon function. atrophy  G,H. ThiThe advantages of mouse models to leverage the genetic labeling of cellular components are balanced by the limitations of small size and lack of complexity of the mouse brain. Mouse models are most useful as tools to screen for pathology that may be otherwise hard to distinguish. These findings in mice should facilitate further development of techniques applicable to higher mammals and humans, and interpretation of clinically applicable techniques such as white matter microstructure effects identified by MRI DTI. The devices available to model blast exposures increase reproducibility by reducing experimental variables and so do not fully replicate the human experience. In the broadest sense, the blast field overall is limited by the need for further studies to more fully establish the human neuropathology of TBI associated with blast exposure.Thy1-YFP-16 mice detected axon damage in white matter tracts across multiple brain regions. NG2CreER;mTmG mice detected new myelin synthesis and provided the ability to visualize the neurovascular-pericyte unit. Taken together, this study demonstrates that fluorescent reporter mice are effective tools for evaluating cellular pathology of blast-induced neurotrauma and that repetitive low-level blast exposures produce infrequent but significant axon damage along with neuroinflammation in white matter.Evaluating effects of low-level repetitive blast exposure is challenging due to potentially subtle and diffuse pathology involving multiple cell types and tissues.


Social isolation, logistical challenges, and limited access to mental health providers who accept Medicare contribute to older adults having a higher risk for untreated depression. Primary care providers (PCPs) are strained due to time demands and lack of training in behavioral activation and similar therapies. This study was designed to reduce depression in older adult primary care patients without burdening their PCPs. We evaluated whether outpatients age 65+ with mild to moderate self-reported depressive symptoms (measured by PHQ-8) benefited from a brief evidence-informed program, the Positive Experience Project(PEP). This is derived from the tenets and practices of both behavioral activation and the “tiny habits” program. The former emphasizes the value of increasing engagement in everyday positive activities to lift mood; the latter provides a method to enhance success by encouraging patients to link these activities with existing habitual behaviors, and to celebrate completion. A script was written for each of four 30-minute sessions conducted in small groups using a telehealth platform. An analysis of patients of the first author yielded 50 eligible patients. 8 were invited to participate and 7 did. The group’s mean PHQ-8 score pre-intervention was 10.1; post-intervention (4 weeks later), the average was 6.1 (P=.039). The use of “scripts” that guide the PCP through the visit enhances adoption. And PCPs can bill for these 30-minute sessions, making it feasible to help patients receive treatment for their depression. These promising results are currently being replicated by additional PCPs and their data will be included in the poster presentation.


Perfusion of dopamine (100 μM) in the extracellular solution significantly reduced peak transient Na+ currents by approximately 20% of control. A decrease in Na+ current amplitude was also seen with extracellular application of the D2 dopamine receptor agonist quinpirole, whereas the D2 receptor antagonist eticlopride largely abolished the response to dopamine. Inclusion of the phosphatase inhibitor okadaic acid in the patch electrode solution occluded the response to dopamine. The reduction in calyx sodium current in response to dopamine suggests efferent signaling through D2 dopaminergic receptors may occur via common mechanisms to decrease excitability in inner ear afferents.Inner ear hair cells form synapses with afferent terminals and afferent neurons carry signals as action potentials to the central nervous system. Efferent neurons have their origins in the brainstem and some make synaptic contact with afferent dendrites beneath hair cells. Several neurotransmitters have been identified that may be released from efferent terminals to modulate afferent activity. Dopamine is a candidate efferent neurotransmitter in both the vestibular and auditory systems. Within the cochlea, activation of dopamine receptors may reduce excitotoxicity at the inner hair cell synapse via a direct effect of dopamine on afferent terminals. Here we investigated the effect of dopamine on sodium currents in acutely dissociated vestibular afferent calyces to determine if dopaminergic signaling could also modulate vestibular responses. Calyx terminals were isolated along with their accompanying type I hair cells from the cristae of gerbils (P15-33) and whole cell patch clamp recordings performed. Large transient sodium currents were present in all isolated calyces; compared to data from crista slices, resurgent Na Intraperitoneal injections of ketamine (70 mg/kg) and xylazine (3 mg/kg) in sterile saline were used to induce anesthesia with supplemental doses given when necessary. Following decapitation the cristae of the semicircular canals were dissected out and immersed in Leibovitz’s L-15 medium . Procedures were in accordance with NIH guidelines and protocols approved by the University of Colorado’s Institutional Animal Care and Use Committee.Mongolian gerbils  and differential interference contrast (DIC) optics on an Olympus microscope (BX51). Isolated cell pairs of single type I hair cells enveloped by calyx terminals or calyces within slices were selected for recordings. Electrodes were pulled from electrode glass  on a multistep horizontal puller  and electrode tips were polished by heat using a Narishige MF 830 microforge . SYLGARD 18  was applied close to the tip of each electrode to reduce stray capacitance.+ currents (+ currents the electrode solution contained (in mM): KF (115), KCl (10), NaCl (2), HEPES (10), D-glucose (3), MgCl2 (2), EGTA (10), pH 7.4 adjusted with KOH (27 mM). For all other experiments the electrode solution contained (in mM): CsF (120), CsCl (10), NaCl (2), HEPES (10), D-glucose (3), MgCl2 (2), EGTA (10), pH 7.4 adjusted with CsOH (24 mM). To further isolate Na+ currents from other conductances, recordings for investigating resurgent currents (INaR) were obtained in an extracellular solution containing in: (mM): NaCl (120), CsCl (5.4), MgCl2 (2.5), tetraethylammonium Cl (30), CaCl2 (1.3), glucose (10), HEPES (5). For all other experiments the external solution was Leibovitz’s L-15 medium.Calyx terminals express several different types of voltage-gated conductances including Nacurrents . For expNaT) and resurgent (INaR) Na+ currents. Data were acquired using pCLAMP 10 software with a patch amplifier  connected to a PC via an AD converter . Signals were low-pass filtered online at 5 or 10 kHz and sampled at 20–50 kHz. Correction for liquid junction potentials was made in the analysis.Electrode resistance was 2.3–5 MΩ prior to seal formation on calyces. Recordings were made at room temperature (21 – 24°C) with voltage protocols designed to probe for transient  at a flow rate of 0.5-1.0 ml/min. Drugs were typically perfused for a minimum of 4-9 min before measurements of drug effects on Na+ currents were taken. The length of the wash (return to control solution) was between 6 and 10 min.Dopamine was dissolved in deionized water or L-15 on each experimental day to make a 10 mM stock solution which was then added to L-15 for a final concentration of 100 μM. In experiments with Cst-tests.Clampfit 10 (Molecular Devices) and Sigmaplot 11  were used to analyze voltage clamp data. Mean values ± SD are reported and values were compared with paired + channels are a target for neuromodulation in many cell types. In cochlear afferent neurons transient Na+ currents are decreased by the application of dopamine  was measured during perfusion of 100 μM dopamine in a P21 isolated calyx  and decreased markedly in the presence of dopamine shortly after the perfusion onset. K+ current amplitude was measured at the end of 40 ms voltage steps to +20 mV and remained relatively unchanged throughout the course of dopamine application. In a group of calyces (ages P21-27) mean IK was 2,161 ± 1,501 pA in control conditions, which was not significantly different from the mean value of 1,976 ± 1,454 pA in dopamine . These experiments suggest that the effect of dopamine is specific to Na+ currents in calyx terminals.To test if dopamine affected voltage-dependent outward Ked calyx . The stacordings . The pea+ currents were not significantly affected by dopamine, we performed additional experiments using Cs+ in the patch electrode solution to inhibit voltage-dependent K+ currents. In the experiments with internal Cs+ outward currents were greatly reduced . The effect of dopamine was tested in four calyces and perfusion with 100 μM dopamine reversibly reduced peak INaT at the test step of –40 mV as shown for one cell in + current amplitude following dopamine exposure and subsequent washout is shown for cells with either K+ or Cs+ as the major cation in the patch electrode solution in + currents were measured at –40 mV and averaged –2,151 ± 1,685 pA in control; following dopamine perfusion mean peak current was reduced to –1,732 ± 1,718 pA (n = 9 cells). The decrease in amplitude was significant and a partial or complete washout was observed in all but two cells on return to normal extracellular solution  and five receptor subtypes (D1-D5) have been described . Based o% n = 6, . Althougdopamine . Eticlop+ current through a change in the phosphorylation state of Na+ channels and inhibition of phosphatase might be expected to produce an inhibition of Na+ currents analogous to activation of dopamine receptors. We therefore tested the effect of okadaic acid, a known inhibitor of phosphatase 1 and 2A, by perfusing cells with extracellular 100 nM okadaic acid. However, we saw no consistent change in amplitude of INa in 3 cells tested with external perfusion of okadaic acid. Next we studied the effect of including 100 nM okadaic acid in the patch electrode solution followed by extracellular dopamine perfusion  were abolished by tetrodotoxin (TTX) (NaR) and persistent currents (INaP), which occurred frequently, but contributed much smaller components of the TTX-sensitive Na+ current than INaT  was observed in greater than half of calyces studied in crista slices at ages P13 and older , INaR was only detected in 2 out of 15 isolated calyces  . We subshan INaT . Dopamin neurons , but we  channel . Calycesnel  in auditory and vestibular afferents, where it acts to shift the activation to more positive potentials and increase the degree of current available around the resting potential  that mediate the upward phase of the action potential. In addition to INaT, resurgent (INaR) and persistent Na+ currents also manifest in certain cell types  and several of these are expressed in vestibular ganglion neurons (+ currents (NaR and INaT were detected together in the majority of mature calyx terminals in crista slices (NaT was present in all isolated calyces studied here, but INaR was rarely observed. In both vestibular calyces and spiral ganglion neurons INaR expression increased with postnatal development suggesting a role for this current in afferent firing in adult inner ear epithelia (NaR expression (NaT, Ih and a variety of K+ currents (NaR generation. Spike generation likely occurs in afferent terminals at the initial unmyelinated segment close to hair cells (+ channel subtypes remains to be determined.Voltage-gated Nall types . Dopamincurrents . Large aodotoxin , 2020. N neurons . Nav1.6 currents . A specia slices . In contpithelia . Navβ4 spression , were depression  and immupression . Althougcurrents , these sir cells , but the+ channels.Vestibular hair cells and inner hair cells make synapses with afferent terminals to convey sound and balance information to the brain as action potentials, but the auditory and vestibular systems also receive extensive efferent input from the brain. Outer hair cells and type II hair cells are contacted directly by efferent terminals, but efferent fibers also terminate on unmyelinated vestibular calyx terminals and auditory afferents postsynaptic to inner hair cells. The strategic placement of efferent synapses in the initial segment zone of the axon and calyx outer face could allow modulation of afferent firing close to the site of action potential initiation through receptor-mediated influence on current components mediated through voltage-gated ion Na2+ influx through α9-containing nicotinic receptors activates SK channels producing a hyperpolarization of type II vestibular hair cells and a decrease in membrane resistance and hair cell sensitivity (+ currents in calyx afferents of turtle and rat crista (B receptor-mediated inhibition of K+ channels (+ currents in vestibular hair cells and afferents, but direct effects of potential neuromodulators on Na+ channels linked to firing have not been reported until now. Our data suggest dopamine release could impact firing in vestibular afferents through activation of D2 receptors and associated intracellular signaling mechanisms resulting in Na+ channel inhibition within calyx terminals. As described in cochlear afferents, dopamine-mediated reduction of INaT would result in a decrease in firing in vestibular calyx-bearing neurons.Gerbil cristae receive substantial efferent innervation with distinct efferent groups projecting to ipsilateral and contralateral cristae . Althougsitivity , 2020. Asitivity . Converst crista . Other tt crista . In rat channels . TherefoDopaminergic efferents are present in the cochlea and may also be present in vestibular epithelia. Tyrosine hydroxylase is required for the synthesis of dopamine and + current inhibition and the involvement of other dopamine receptor subtypes remains to be elucidated. Disruption to dopaminergic efferent pathways could be associated with auditory and vestibular dysfunction.Dopamine may operate as a neuroactive substance at the hair cell/afferent synapse in mammalian auditory and vestibular systems. Through a reduction in sodium channel activity via changes in phosphorylation, dopamine released from efferents may reduce action potential firing in both auditory and vestibular afferents. In the cochlea, dopamine appears to exert its inhibitory effect on afferent firing via two distinct dopaminergic signaling pathways. Our data support a role for D2 receptors in NaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by University of Colorado Denver Institutional Animal Care and Use Committee.FM and KR designed the experiments, performed the research experiments, analyzed the data, and prepared the figures. KR wrote the first draft of the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Perioperative interventions produce substantial biologic perturbations which are associated with the risk of recurrence after cancer surgery. The changes of tumor microenvironment caused by anesthetic drugs received increasing attention. Till now, it’s still unclear whether or not anesthetic drugs may exert positive or negative impact on cancer outcomes after surgery. Breast cancer is the most common tumor and the leading cause of cancer deaths in women. Propofol and sevoflurane are respectively the most commonly used intravenous and inhaled anesthetics. Debates regarding which of the two most commonly used anesthetics may relatively contribute to the recurrence and metastasis vulnerability of breast cancer postoperatively remain. This review aimed to provide a comprehensive view about the effect of propofol versus sevoflurane on the prognosis of breast cancer obtained from pre-clinical studies and clinical studies. Laboratory and animal studies have demonstrated that sevoflurane may enhance the recurrence and metastasis of breast cancer, while propofol is more likely to reduce the activity of breast cancer cells by attenuating the suppression of the immune system, promoting tumor cells apoptosis, and through other direct anti-tumor effects. However, retrospective clinical studies have shown contradictory results about the effects of propofol and sevoflurane on long-term survival in breast cancer patients. Furthermore, recent prospective studies did not identify significant differences between propofol and sevoflurane in breast cancer metastasis and recurrence. Therefore, more preclinical studies and randomized controlled studies are needed to guide the choice of anesthetics for breast cancer patients. Breast cancer is the most commonly diagnosed malignant tumor and the leading cause of cancer-related death among females. It was estimated that there were more than 2 million new cases and 0.63 million cancer related deaths worldwide in the single year of 2018 , 5. The There have been increasing concerns that perioperative substantial biologic perturbations will increase the risk of recurrence after cancer surgery . On the This review aimed to compare the effects of propofol versus sevoflurane on immune system, breast cancer cells and patient long-term outcomes observed from pre-clinical studies and clinical studies. We searched PubMed database with search terms (“propofol” or “sevoflurane”) and (“breast cancer” or “breast tumor”) on Sept. 30, 2021 to obtain the literatures in this review, and only the articles written in English were included.+Th1, CD8+CTL (cytotoxic T lymphocyte), and cytokines including tumor necrosis factor-α (TNF-α), interferon-α, interferon-β, interferon-γ and interleukin-12(IL-12) are the primary factors to recognize and eliminate cancer cells  cells, CD4er cells . If the In addition to the host’s anti-tumor immunity, tumor cells also produce mediators that fight against host immunity in order to promote their own growth. The cytokines such as VEGF and transforming growth factor-β (TGF-β) which are produced by tumor cells can induce immunosuppressive effects , 19. SomSevoflurane is the most popular volatile anesthetics due to the advantages of fast induction, small respiratory tract stimulation, fast absorption and clearance, less circulation disturbance. Propofol, a kind of alkyl acid short acting anesthetics, is the most commonly used intravenous anesthetic. Laboratory researches have shown that propofol-based intravenous anesthesia and sevoflurane-based inhalation anesthesia may have different effects on breast cancer immune microenvironment.+ CTL and CD4+ Th1 cells are the important weapons to fight against cancer cells  . Increasin vitro to investigate the effect of anesthetics on breast cancer cell proliferation, migration and apoptosis , progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), the diverse subtypes are stratified into four major molecular subtypes namely triple negative breast cancer cell, HER2 overexpressing breast cancer cell, Luminal A breast cancer cell and Luminal B breast cancer cell. Triple negative breast cancer cell is ER-/PR-/HER2-; HER2 overexpressing breast cancer cell is ER-/PR-/HER2+; Luminal B breast cancer cell is ER+ and/or PR+/HER2+; Luminal A breast cancer cell is ER+ and/or PR+/HER2-. In recent years, the potential impact of different general anesthetics on tumor prognosis has garnered particular attention. Different breast cancer cell lines were cultured poptosis .in vitro study investigated the effect of sevoflurane on breast cancer cell proliferation, migration and invasion  has been identified to have the property of promoting tumor cells migration , and hasn of 4mM . Patricin of 4mM . In addin of 4mM , 55. Then of 4mM  and has n of 4mM . HIF-1 an of 4mM . An expen of 4mM . There an of 4mM .via activating STAT3 and infiltrating CD11b+ cells as compared to propofol  provide a favorable microenvironment for tumorigenesis by digesting extracellular matrix components. MMPs also release pro-cancer factors from the extracellular matrix to promote tumor cell migration . The levpropofol . Generalpropofol , 67. A ppropofol .2+, a kind of second messenger, plays a key role in numerous cellular processes including cell proliferation and apoptosis  with no statistical significance. Then, a retrospective study from Korea compared the recurrence-free survival and overall survival between propofol and sevoflurane groups in patients after modified radical mastectomy  are badly needed. Other anesthetic drugs and anesthetic techniques are also of concern in breast cancer surgery. Due to the analgesic properties, opioids are widely used during breast cancer surgery. Some laboratory studies showed that opioids inhibit cell-mediated immunity , reduce Overall, pre-clinical studies and retrospective clinical studies comparing the potential benefits of intravenous propofol over inhalational anesthetics for breast cancer lack consistency. A few current randomized controlled studies suggest that the two anesthetics have similar effects on breast cancer recurrence and metastasis. However, a definite conclusion regarding which anesthetic may have more favorable long-term effects on breast cancer recurrence and metastasis cannot be reached largely due to the lack of multicenter or multi-countries large sample clinical trials.So far, the effect of different anesthetics or anesthesia techniques on the prognosis of postoperative breast cancer has not been determined. Further investigations should be implemented to explore the mechanisms of anesthetics on breast cancer cells and immune microenvironment. Meanwhile, large sample, multi-center prospective clinical study involving different subtype of breast cancer, different tumor staging should also be conducted. Only a clear understanding of the relationship between anesthetics and breast cancer can improve the prognosis of patients from the perspective of anesthesiologists.YL and PF had the original idea of the manuscript. PF, JZ, and YL reviewed the literature and drafted the article. ZX and XL revised manuscript and provided suggestions for improvement. All authors contributed to the article and approved the submitted version.The author’s study was supported by Advanced Projects of Innovation Program for the Selected Returned Overseas Chinese Scholars, Anhui Province (2020LCX022); National Natural Science Foundation of China (81770295); Bethune Charity Fund project (BCF-RF-WSQZTZJ-202011-057); Young Scholars of Wan Jiang in Anhui Province.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.