Protocol for highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence in rodents.

We present a protocol to induce Cre-dependent transgene expression in specific cell types in the rat brain, suppressing a leak expression in off-target cells, by using a flip-excision switch system with a unilateral spacer sequence. We describe steps for construction of transfer plasmids, preparation of adeno-associated viral vectors, intracranial injection, and detection of transgene expression. Our protocol provides a useful strategy for a better understanding of the structure and function of specific cell types in the complex neural circuit. For complete details on the use and execution of this protocol, please refer to Matsushita et al.1.

Anatomical identification of a corticocortical top-down recipient inhibitory circuitry by enhancer-restricted transsynaptic tracing.

Despite the importance of postsynaptic inhibitory circuitry targeted by mid/long-range projections (e.g., top-down projections) in cognitive functions, its anatomical properties, such as laminar profile and neuron type, are poorly understood owing to the lack of efficient tracing methods. To this end, we developed a method that combines conventional adeno-associated virus (AAV)-mediated transsynaptic tracing with a distal-less homeobox (Dlx) enhancer-restricted expression system to label postsynaptic inhibitory neurons. We called this method "Dlx enhancer-restricted Interneuron-SpECific transsynaptic Tracing" (DISECT). We applied DISECT to a top-down corticocortical circuit from the secondary motor cortex (M2) to the primary somatosensory cortex (S1) in wild-type mice. First, we injected AAV1-Cre into the M2, which enabled Cre recombinase expression in M2-input recipient S1 neurons. Second, we injected AAV1-hDlx-flex-green fluorescent protein (GFP) into the S1 to transduce GFP into the postsynaptic inhibitory neurons in a Cre-dependent manner. We succeeded in exclusively labeling the recipient inhibitory neurons in the S1. Laminar profile analysis of the neurons labeled via DISECT indicated that the M2-input recipient inhibitory neurons were distributed in the superficial and deep layers of the S1. This laminar distribution was aligned with the laminar density of axons projecting from the M2. We further classified the labeled neuron types using immunohistochemistry and in situ hybridization. This post hoc classification revealed that the dominant top-down M2-input recipient neuron types were somatostatin-expressing neurons in the superficial layers and parvalbumin-expressing neurons in the deep layers. These results demonstrate that DISECT enables the investigation of multiple anatomical properties of the postsynaptic inhibitory circuitry.

Highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence.

The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections.

For achieving retrograde gene transfer, we have so far developed two types of lentiviral vectors pseudotyped with fusion envelope glycoprotein, termed HiRet vector and NeuRet vector, consisting of distinct combinations of rabies virus and vesicular stomatitis virus glycoproteins. In the present study, we compared the patterns of retrograde transgene expression for the HiRet vs. NeuRet vectors by testing the cortical input system. These vectors were injected into the motor cortex in rats, marmosets, and macaques, and the distributions of retrograde labels were investigated in the cortex and thalamus. Our histological analysis revealed that the NeuRet vector generally exhibits a higher efficiency of retrograde gene transfer than the HiRet vector, though its capacity of retrograde transgene expression in the macaque brain is unexpectedly low, especially in terms of the intracortical connections, as compared to the rat and marmoset brains. It was also demonstrated that the NeuRet but not the HiRet vector displays sufficiently high neuron specificity and causes no marked inflammatory/immune responses at the vector injection sites in the primate (marmoset and macaque) brains. The present results indicate that the retrograde transgene efficiency of the NeuRet vector varies depending not only on the species but also on the input projections.

Optimization of the delivery of molecules into lymph nodes using a lymphatic drug delivery system with ultrasound.

Conventional treatment for lymph node (LN) metastasis such as systemic chemotherapy have notable disadvantages that lead to the development of unwanted effects. Previously, we have reported the lymphatic administration of drugs into metastatic LNs using a lymphatic drug delivery system (LDDS). However, prior studies of the LDDS have not attempted to optimize the conditions for efficient drug delivery. Here, we investigated the influence of several factors on the efficiency of drug delivery by a LDDS in conjunction with ultrasound (US). First, the effect of the injection rate on delivery efficiency was evaluated. Fluorescent molecules injected into an upstream LN were delivered more effectively into a downstream LN when a lower injection rate was used. Second, the influence of molecular weight on drug delivery efficiency was determined. We found that molecules with a molecular weight >10,000 were poorly delivered into the LN. Finally, we assessed whether the administration route affected the delivery efficiency. We found that the delivery efficiency was higher when molecules were administered into an upstream LN that was close to the target LN. These findings revealed the importance of a drug's physical properties if it is to be administered by LDDS to treat LN metastasis.

Intranodal pressure of a metastatic lymph node reflects the response to lymphatic drug delivery system.

Cancer metastasis to lymph nodes (LNs) almost certainly contributes to distant metastasis. Elevation of LN internal pressure (intranodal pressure, INP) during tumor proliferation is associated with a poor prognosis for patients. We have previously reported that a lymphatic drug delivery system (LDDS) allows the direct delivery of anticancer drugs into the lymphatic system and is a promising treatment strategy for early-stage LN metastasis. However, methods for evaluating the treatment effects have not been established. Here, we used a mouse model of MXH10/Mo-lpr/lpr, which develops a systemic swelling of LNs, and murine malignant fibrous histiocytoma-like (KM-Luc/GFP) cells or murine breast cancer (FM3A-Luc) cells inoculated into the subiliac LN of mice to produce a tumor-bearing LN model. The changes in INP during intranodal tumor progression and after treatment with cis-dichlorodiammineplatinum(II) (CDDP) using an LDDS were measured. We found that tumor progression was associated with an increase in INP that occurred independently of LN volume changes. The elevation in INP was suppressed by CDDP treatment with the LDDS when intranodal tumor progression was significantly inhibited. These findings indicate that INP is a useful parameter for monitoring the therapeutic effect in patients with LN metastasis who have been given drugs using an LDDS, which will serve to manage cancer metastasis treatment and contribute to an improved quality of life for cancer patients.

Dopaminergic Signaling in the Nucleus Accumbens Modulates Stress-Coping Strategies during Inescapable Stress.

Maladaptation to stress is a critical risk factor in stress-related disorders, such as major depression and post-traumatic stress disorder (PTSD). Dopamine signaling in the nucleus accumbens (NAc) has been shown to modulate behavior by reinforcing learning and evading aversive stimuli, which are important for the survival of animals under environmental challenges such as stress. However, the mechanisms through which dopaminergic transmission responds to stressful events and subsequently regulates its downstream neuronal activity during stress remain unknown. To investigate how dopamine signaling modulates stress-coping behavior, we measured the subsecond fluctuation of extracellular dopamine concentration and pH using fast scanning cyclic voltammetry (FSCV) in the NAc, a postsynaptic target of midbrain dopaminergic neurons, in male mice engaged in a tail suspension test (TST). The results revealed a transient decrease in dopamine concentration and an increase in pH levels when the animals changed behaviors, from being immobile to struggling. Interestingly, optogenetic inhibition of dopamine release in NAc, potentiated the struggling behavior in animals under the TST. We then addressed the causal relationship of such a dopaminergic transmission with behavioral alterations by knocking out both the dopamine receptors, i.e., D1 and D2, in the NAc using viral vector-mediated genome editing. Behavioral analyses revealed that male D1 knock-out mice showed significantly more struggling bouts and longer struggling durations during the TST, while male D2 knock-out mice did not. Our results therefore indicate that D1 dopaminergic signaling in the NAc plays a pivotal role in the modulation of stress-coping behaviors in animals under tail suspension stress.SIGNIFICANCE STATEMENT The tail suspension test (TST) has been widely used as a despair-based behavioral assessment to screen the antidepressant so long. Despite its prevalence in the animal studies, the neural substrate underlying the changes of behavior during the test remains unclear. This study provides an evidence for a role of dopaminergic transmission in the modulation of stress-coping behavior during the TST, a despair test widely used to screen the antidepressants in rodents. Taking into consideration the fact that the dopamine metabolism is upregulated by almost all antidepressants, a part of which acts directly on the dopaminergic transmission, current results would uncover the molecular mechanism through which the dopaminergic signaling mediates antidepressant effect with facilitation of the recovery from the despair-like behavior in the TST.

Pseudotyped lentiviral vectors for tract-targeting and application for the functional control of selective neural circuits.

A lentiviral vector strategy for efficient gene transfer through retrograde axonal transport provides a powerful approach for studying the neural circuit mechanisms that mediate higher level functions of the central nervous system. Pseudotyping of human immunodeficiency virus type-1 with different types of fusion glycoproteins (FuGs), which are composed of segments of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G), enhances the efficiency of retrograde gene transfer in both rodent and non-human primate brains. These pseudotyped lentiviral vectors are classified into two groups, highly efficient retrograde gene transfer (HiRet) and neuron-specific retrograde gene transfer (NeuRet) vectors, based on their properties of gene transduction. Combinatorial use of the pseudotyped vectors with various molecular tools for manipulating neural circuit functions (such as the cell targeting, synaptic silencing, and optogenetic or chemogenetic approaches) enables us to control the function of specific neural circuits, thus leading to a deeper understanding of the mechanism underlying various nervous system functions.

Use of a Lymphatic Drug Delivery System and Sonoporation to Target Malignant Metastatic Breast Cancer Cells Proliferating in the Marginal Sinuses.

Lymph node (LN) metastasis through the lymphatic network is a major route for cancer dissemination. Tumor cells reach the marginal sinuses of LNs via afferent lymphatic vessels (LVs) and form metastatic lesions that lead to distant metastasis. Thus, targeting of metastatic cells in the marginal sinuses could improve cancer treatment outcomes. Here, we investigated whether lymphatic administration of a drug combined with sonoporation could be used to treat a LN containing proliferating murine FM3A breast cancer cells, which are highly invasive, in its marginal sinus. First, we used contrast-enhanced high-frequency ultrasound and histopathology to analyze the structure of LVs in MXH10/Mo-lpr/lpr mice, which exhibit systemic lymphadenopathy. We found that contrast agent injected into the subiliac LN flowed into the marginal sinus of the proper axillary LN (PALN) and reached the cortex. Next, we examined the anti-tumor effects of our proposed technique. We found that a strong anti-tumor effect was achieved by lymphatic administration of doxorubicin and sonoporation. Furthermore, our proposed method prevented tumor cells in the marginal sinus from invading the parenchyma of the PALN and resulted in tumor necrosis. We conclude that lymphatic administration of a drug combined with sonoporation could exert a curative effect in LNs containing metastatic cells in their marginal sinuses.

Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression.

The effects of autonomic innervation of tumors on tumor growth remain unclear. Here we developed a series of genetic techniques to manipulate autonomic innervation in a tumor- and fiber-type-specific manner in mice with human breast cancer xenografts and in rats with chemically induced breast tumors. Breast cancer growth and progression were accelerated following stimulation of sympathetic nerves in tumors, but were reduced following stimulation of parasympathetic nerves. Tumor-specific sympathetic denervation suppressed tumor growth and downregulated the expression of immune checkpoint molecules (programed death-1 (PD-1), programed death ligand-1 (PD-L1), and FOXP3) to a greater extent than with pharmacological α- or ß-adrenergic receptor blockers. Genetically induced simulation of parasympathetic innervation of tumors decreased PD-1 and PD-L1 expression. In humans, a retrospective analysis of breast cancer specimens from 29 patients revealed that increased sympathetic and decreased parasympathetic nerve density in tumors were associated with poor clinical outcomes and correlated with higher expression of immune checkpoint molecules. These findings suggest that autonomic innervation of tumors regulates breast cancer progression.

Enhancement of the transduction efficiency of a lentiviral vector for neuron-specific retrograde gene delivery through the point mutation of fusion glycoprotein type E.

BACKGROUND: Pseudotyping of a lentiviral vector with fusion glycoproteins composed of rabies virus glycoprotein (RVG) and vesicular stomatitis virus glycoprotein (VSVG) segments achieves high gene transfer efficiency through retrograde transport in the nervous system. In our previous study, we determined the junction of RVG/VSVG segments of glycoproteins that enhances the transduction efficiency of the neuron-specific retrograde gene transfer (NeuRet) vector (termed fusion glycoprotein type E or FuG-E). NEW METHOD: We aimed to optimize the amino acid residue at position 440 in the membrane-proximal region of FuG-E to improve the efficiency of retrograde gene transfer in the brain. RESULTS: We constructed variants of FuG-E with 18 kinds of single amino acid substitutions at residue 440 to generate lentiviral vectors pseudotyped with these variants, and tested in vivo gene transfer of the vectors in the mouse brain. The FuG-E (P440E) variant, in which proline was substituted by glutamate at residue 440 in FuG-E, showed the greatest retrograde gene transfer efficiency in the brain, bearing the property of the NeuRet vector. The FuG-E (P440E) pseudotype also displayed efficient retrograde gene transfer in the common marmoset brain. COMPARISON WITH EXISTING METHODS: The NeuRet vector with the FuG-E (P440E) variant demonstrated higher retrograde gene transfer efficiency into different neural pathways compared with the parental FuG-E vector. CONCLUSIONS: The FuG-E (P440E) pseudotype provides a powerful tool to investigate neural circuit mechanisms underlying various brain functions and for gene therapy trials of neurological and neurodegenerative diseases.

Anatomical Evidence for a Direct Projection from Purkinje Cells in the Mouse Cerebellar Vermis to Medial Parabrachial Nucleus.

Cerebellar malformations cause changes to the sleep-wake cycle, resulting in sleep disturbance. However, it is unclear how the cerebellum contributes to the sleep-wake cycle. To examine the neural connections between the cerebellum and the nuclei involved in the sleep-wake cycle, we investigated the axonal projections of Purkinje cells in the mouse posterior vermis by using an adeno-associated virus (AAV) vector (serotype rh10) as an anterograde tracer. When an AAV vector expressing humanized renilla green fluorescent protein was injected into the cerebellar lobule IX, hrGFP and synaptophysin double-positive axonal terminals were observed in the region of medial parabrachial nucleus (MPB). The MPB is involved in the phase transition from rapid eye movement (REM) sleep to Non-REM sleep and vice versa, and the cardiovascular and respiratory responses. The hrGFP-positive axons from lobule IX went through the ventral spinocerebellar tract and finally reached the MPB. By contrast, when the AAV vector was injected into cerebellar lobule VI, no hrGFP-positive axons were observed in the MPB. To examine neurons projecting to the MPB, we unilaterally injected Fast Blue and AAV vector (retrograde serotype, rAAV2-retro) as retrograde tracers into the MPB. The cerebellar Purkinje cells in lobules VIII-X on the ipsilateral side of the Fast Blue-injected MPB were retrogradely labeled by Fast Blue and AAV vector (retrograde serotype), but no retrograde-labeled Purkinje cells were observed in lobules VI-VII and the cerebellar hemispheres. These results indicated that Purkinje cells in lobules VIII-X directly project their axons to the ipsilateral MPB but not lobules VI-VII. The direct connection between lobules VIII-X and the MPB suggests that the cerebellum participates in the neural network controlling the sleep-wake cycle, and cardiovascular and respiratory responses, by modulating the physiological function of the MPB.

Neuronal cytoskeletal gene dysregulation and mechanical hypersensitivity in a rat model of Rett syndrome.

Children with Rett syndrome show abnormal cutaneous sensitivity. The precise nature of sensory abnormalities and underlying molecular mechanisms remain largely unknown. Rats with methyl-CpG binding protein 2 (MeCP2) mutation, characteristic of Rett syndrome, show hypersensitivity to pressure and cold, but hyposensitivity to heat. They also show cutaneous hyperinnervation by nonpeptidergic sensory axons, which include subpopulations encoding noxious mechanical and cold stimuli, whereas peptidergic thermosensory innervation is reduced. MeCP2 knockdown confined to dorsal root ganglion sensory neurons replicated this phenotype in vivo, and cultured MeCP2-deficient ganglion neurons showed augmented axonogenesis. Transcriptome analysis revealed dysregulation of genes associated with cytoskeletal dynamics, particularly those controlling actin polymerization and focal-adhesion formation necessary for axon growth and mechanosensory transduction. Down-regulation of these genes by topoisomerase inhibition prevented abnormal axon sprouting. We identified eight key affected genes controlling actin signaling and adhesion formation, including members of the Arhgap, Tiam, and cadherin families. Simultaneous virally mediated knockdown of these genes in Rett rats prevented sensory hyperinnervation and reversed mechanical hypersensitivity, indicating a causal role in abnormal outgrowth and sensitivity. Thus, MeCP2 regulates ganglion neuronal genes controlling cytoskeletal dynamics, which in turn determines axon outgrowth and mechanosensory function and may contribute to altered pain sensitivity in Rett syndrome.

The use of an optimized chimeric envelope glycoprotein enhances the efficiency of retrograde gene transfer of a pseudotyped lentiviral vector in the primate brain.

Lentiviral vectors have been used not only for various basic research experiments, but also for a wide range of gene therapy trials in animal models. The development of a pseudotyped lentiviral vector with the property of retrograde infection allows us to introduce foreign genes into neurons that are localized in regions innervating the site of vector injection. Here, we report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra. Quantitative analysis revealed that in all regions, the number of neurons retrogradely transduced with the FuG-E vector was larger than in the FuG-C vector injection case. It was also confirmed that the FuG-E vector displayed explicit neuronal specificity to the same extent as the FuG-C vector. This vector might promote approaches to pathway-selective gene manipulation and provide a powerful tool for effective gene therapy trials against neurological disorders through enhanced retrograde delivery.

Survival of corticostriatal neurons by Rho/Rho-kinase signaling pathway.

Developing cortical neurons undergo a number of sequential developmental events including neuronal survival/apoptosis, and the molecular mechanism underlying each characteristic process has been studied in detail. However, the survival pathway of cortical neurons at mature stages remains largely uninvestigated. We herein focused on mature corticostriatal neurons because of their important roles in various higher brain functions and the spectrum of neurological and neuropsychiatric disorders. The small GTPase Rho is known to control diverse and essential cellular functions through some effector molecules, including Rho-kinase, during neural development. In the present study, we investigated the role of Rho signaling through Rho-kinase in the survival of corticostriatal neurons. We performed the conditional expression of Clostridium botulinum C3 ADP-ribosyltransferase (C3 transferase) or dominant-negative form for Rho-kinase (Rho-K DN), a well-known inhibitor of Rho or Rho-kinase, respectively, in corticostriatal neurons using a dual viral vector approach combining a neuron-specific retrograde gene transfer lentiviral vector and an adeno-associated virus vector. C3 transferase markedly decreased the number of corticostriatal neurons, which was attributed to caspase-3-dependent enhanced apoptosis. In addition, Rho-K DN produced phenotypic defects similar to those caused by C3 transferase. These results indicate that the Rho/Rho-kinase signaling pathway plays a crucial role in the survival of corticostriatal neurons.

Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders.

A Novel Treatment Method for Lymph Node Metastasis Using a Lymphatic Drug Delivery System with Nano/Microbubbles and Ultrasound.

Chemotherapy based on hematogenous administration of drugs to lymph nodes (LNs) located outside the surgically resected area shows limited tissue selectivity and inadequate response rates, resulting in poor prognosis. Here, we demonstrate proof of concept for a lymphatic drug delivery system using nano/microbubbles (NMBs) and ultrasound (US) to achieve sonoporation in LNs located outside the dissection area. First, we demonstrated the in vitro effectiveness of doxorubicin (Dox) delivered into three different tumor cell lines by sonoporation. Sonoporation increased the Dox autofluorescence signal and resulted in a subsequent decrease in cell viability. Next, we verified the antitumor effects of Dox in vivo using MXH10/Mo-lpr/lpr mice that exhibit systemic lymphadenopathy, with some peripheral LNs reaching 10 mm in diameter. We defined the subiliac LN (SiLN) as the upstream LN within the dissection area, and the proper axillary LN (PALN) as the downstream LN outside the dissection area. Dox and NMBs were injected into the SiLN and delivered to the PALN via lymphatic vessels; the PALN was then exposed to US when it had filled with solution. We found that sonoporation enhanced the intracellular uptake of Dox leading to high cytotoxicity. We also found that sonoporation induced extravasation of Dox from lymphatic endothelia and penetration of Dox into tumor tissues within the PALN. Furthermore, our method inhibited tumor growth and diminished blood vessels in the PALN while avoiding systemic toxic effects of Dox. Our findings indicate that a lymphatic drug delivery system with sonoporation represents a promising method for treating metastatic LNs located outside the dissection area.

Double Virus Vector Infection to the Prefrontal Network of the Macaque Brain.

To precisely understand how higher cognitive functions are implemented in the prefrontal network of the brain, optogenetic and pharmacogenetic methods to manipulate the signal transmission of a specific neural pathway are required. The application of these methods, however, has been mostly restricted to animals other than the primate, which is the best animal model to investigate higher cognitive functions. In this study, we used a double viral vector infection method in the prefrontal network of the macaque brain. This enabled us to express specific constructs into specific neurons that constitute a target pathway without use of germline genetic manipulation. The double-infection technique utilizes two different virus vectors in two monosynaptically connected areas. One is a vector which can locally infect cell bodies of projection neurons (local vector) and the other can retrogradely infect from axon terminals of the same projection neurons (retrograde vector). The retrograde vector incorporates the sequence which encodes Cre recombinase and the local vector incorporates the "Cre-On" FLEX double-floxed sequence in which a reporter protein (mCherry) was encoded. mCherry thus came to be expressed only in doubly infected projection neurons with these vectors. We applied this method to two macaque monkeys and targeted two different pathways in the prefrontal network: The pathway from the lateral prefrontal cortex to the caudate nucleus and the pathway from the lateral prefrontal cortex to the frontal eye field. As a result, mCherry-positive cells were observed in the lateral prefrontal cortex in all of the four injected hemispheres, indicating that the double virus vector transfection is workable in the prefrontal network of the macaque brain.

Communication between lymphatic and venous systems in mice.

The lymphatic system in mice consists of lymphatic vessels and 22 types of lymph nodes. Metastatic tumor cells in the lymphatic system spread to distant organs through the venous system. However, the communication routes between the lymphatic and venous systems have not been fully elucidated. Here, we identify the communication routes between the lymphatic and venous systems in the axillary and subiliac regions of MXH10/Mo-lpr/lpr inbred mice, which develop systemic swelling of lymph nodes up to 10mm in diameter, allowing investigation of the topography of the lymph nodes and lymphatic vessels. Using a gross anatomy dissection approach, the efferent lymphatic vessels of the proper axillary lymph node were shown to communicate with the subclavian vein. Furthermore, we found that the thoracoepigastric vein, which connects the subclavian vein and inferior vena cava, runs adjacent to the subiliac and proper axillary lymph nodes, and receives venous blood from these lymph nodes routed through small branches. The direction of blood flow in the thoracoepigastric vein occurred in two directions in the intermediate region between the proper axillary lymph node and subiliac lymph node; one to the subclavian vein, the other to the inferior vena cava. This paper reveals the anatomy of the communication between the lymphatic and venous systems in the axillary and subiliac regions of the mouse, and provides new insights relevant to the investigation of the mechanisms of lymph node metastasis and cancer immunology, and the development of diagnostic and treatment methods for lymph node metastasis, including drug delivery systems.