Optic Nerve Cerebrospinal Fluid Drained-out via Its Sheath Lymphatic Vessels

Optic Nerve Cerebrospinal Fluid Drained-out via Its Sheath Lymphatic Vessels | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Optic Nerve Cerebrospinal Fluid Drained-out via Its Sheath Lymphatic Vessels Yang Xu, Wenjing Chen, Yuwei Hu, Huan Wang, Min Yan, Yuan Xie, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7083215/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The cerebrospinal fluid (CSF) surrounding the optic nerve (to differentiate with the intracranial CSF, it is named as optic nerve CSF) plays important roles in nutrient exchange, waste removal, and maintenance of optic nerve function. Recent studies have suggested that the optic disc edema observed in astronauts may be linked to the retention of CSF around the optic nerve, and this retention may even be derived from impaired drainage of optic nerve CSF. However, how the optic nerve CSF is drained out remains inconclusive. We speculated that the optic nerve CSF may be drained out through lymphatic vessels of the optic nerve sheath. Here, we found by immunofluorescence and in vivo fluorescence imaging that, in both rats and humans, the optic nerve sheath has lymphatic vessels. Furthermore, we observed that the pressure in the optic nerve CSF is lower than the intracranial pressure, and the optic nerve CSF does not reflux into the intracranial space but is, instead, drained out into the deep cervical lymph nodes (dcLNs) through the lymphatic vessels in the optic nerve sheath. These observations have significant implications for understanding the physiological turnover and drainage pathways of optic nerve CSF. It may also help better understand the pathogenesis underlying spaceflight and Spaceflight Associated Neuro-Ocular Syndrome (SANS). Health sciences/Diseases Health sciences/Medical research Health sciences/Neurology Biological sciences/Neuroscience optic nerve CSF sub-sheath space of the optic nerve lymphatic vessels optic nerve sheath spaceflight associated neuro-ocular syndrome (SANS) intracranial and intraocular pressure difference Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In 2011, Mader et al. first reported the association between spaceflight and Spaceflight Associated Neuro-Ocular Syndrome (SANS), noting that astronauts exhibited optic nerve sheath expansion, optic disc edema, choroidal folds, globe flattening,and hypermetropic axial length changes following long-duration spaceflight[ 1 ]. Subsequent studies have shown that SANS may persist for years in some individuals[ 2 , 3 ]. The currently prevailing hypothesis suggests that these changes are caused by fluid redistribution in the microgravity environment[ 4 – 7 ], where alterations in gravity and hydrostatic pressure impact the transmural pressure in arteries, veins, and lymphatic vessels, leading to changes in local blood flow and lymphatic drainage. However, a critical question remains unresolved: Why do some post-flight changes, such as optic nerve cerebrospinal fluid (CSF) retention and optic nerve sheath expansion showed in magnetic resonance image (MRI), persist even long after astronauts return to Earth? In physiology, the CSF surrounding the optic nerve (to differentiate the with the intracranial CSF, it is named as optic nerve CSF) plays an important role in nutrient exchange, waste removal, and maintenance of optic nerve function.Recently, some studies suggested that, the optic disc edema observed in astronauts may be linked to the retention of the optic nerve CSF[ 6 , 8 , 9 ], and this retention may be anatomically associated with impaired drainage of optic nerve CSF[ 10 ]. Some scholars argue that the elevation in the optic nerve sheath pressure (ONSP) due to microgravity-induced headward fluid shift is the primary hypothesized contributor to SANS[ 11 ]. These hypotheses highlight the urgent need to elucidate the circulation of optic nerve CSF. Traditionally, it has been believed that intracranial CSF flows into and round the optic nerve through the optic canal, and subsequently drains back into the cranial cavity. Although the inflow pathway of optic nerve CSF is widely accepted, controversies remain as to whether it drains back into the intracranial space[ 10 , 12 , 13 ]. Killer et al. argued that it is unlikely for optic nerve CSF to flow back into the cranial cavity against the volume gradient, nor is it probable for bidirectional flow to occur within the optic canal[ 12 ]. Their perspectives are based on clinical evidences[ 14 ]. But, if optic nerve CSF is not drained out through the optic canal, what alternative out-drainage pathway could it be? In past decades, a revolutionary theory proposed that lymphatic vessels within the meninge can drain intracranial CSF out into the deep cervical lymph nodes (dcLNs)[ 15 ]. It is well-known that the optic nerve is an anatomical extension of the central nervous system toward the orbit, surrounded by CSF and its sheath (an extension of the meninge). Thus, we hypothesize that, 1) lymphatic vessels may also exist in the optic nerve sheath, 2) optic nerve CSF may drain via these vessels. To verify these hypotheses, the present study focuses on the drainage pathway of optic nerve CSF.Our results demonstrate that, optic nerve CSF does not flow back into the intracranial space, but instead drains out into the dcLNs via lymphatic vessels in the optic nerve sheath. This discovery has significant implications for elucidating the drainage pathways of optic nerve CSF, and may be helpful for understanding the pathological nature of SANS. Results 1 The pressure in the sub-sheath space of optic nerve is lower than that in the intracranial space In this study, we designed and fabricated a pressure tester (Fig. 1 a and 1 b) to measure the pressure in the sub-sheath space of the optic nerve (PSSON). The calibration of the pressure tester showed a strong linear correlation between the pressure output by the pneumatic pressure gauge and the tester, confirming its accuracy (slope = 1.001 ± 0.001, p < 0.001; intercept = -0.030 ± 0.001, p = 0.967; R² = 1.0000)(Fig. 1 c). Using the pressure tester, we measured the intracranial pressure (ICP) and the PSSON in rabbits (Fig. 2 a and 2 b). The PSSON was measured to be 12.88 ± 2.94 mmH 2 O (n = 12), while the ICP was 20.88 ± 3.75 mmH 2 O. The PSSON was significantly lower than the ICP (n = 12, paired t-test, ****p < 0.0001)(Fig. 2 b). Subsequently, we established an intracranial hypertension model in rabbits by normal saline perfusion. In these rabbits, the PSSON was measured to be 273.7 ± 6.00 mmH 2 O, and the ICP 301.4 ± 1.4 mmH 2 O. Again the PSSON remained significantly lower than the ICP (n = 10, paired t-test, **p < 0.01)(Fig. 2 c). 2 The optic nerve CSF does not flow back into the intracranial space To investigate whether optic nerve CSF flows back into the intracranial space, Alexa Fluor 488 was injected into the sub-sheath space of the right optic nerve in rats. It was found 10–30 min after injection, the fluorescence intensity of Alexa Fluor 488 in the optic nerve CSF progressively decreased, indicating its drainage. Importantly, at each time point, the fluorescence intensity measured from the optic chiasm cystern CSF was markedly lower (Fig. 3 b). These findings strongly suggest that, the optic nerve CSF did not flow back into the intracranial space. 3. The optic nerve CSF flows to dcLNs To study the outflow pathway of the optic nerve CSF, we injected the tracer Qdot655 into the sub-sheath space of the right optic nerve in rats. Ninety minutes later, strong fluorescence signals were detected in the right dcLN, with weaker fluorescence observed in the left dcLN (Fig. 4 b and 4 c). Furthermore, 30 minutes after the injection of Alexa Fluor 488 into the sub-sheath space of the right optic nerve (Fig. 4 d), fluorescence intensity in the dcLN was significantly higher than that in the optic nerve CSF (Fig. 4 e). Collectively, These findings supports the conclusion that optic nerve CSF flows to the dcLNs. 4.Presence of lymphatic vessels in the optic nerve sheath In past decades, a revolutionary theory was proposed, that lymphatic vessels within the meninge can drain intracranial CSF out into the dcLNs [ 14 , 15 , 16 ]. It is well-known that the optic nerve is an anatomical extension of the central nervous system toward the orbit, surrounded by CSF and its sheath (an extension of the meninge). Thus, we hypothesize that, lymphatic vessels may also exist in the optic nerve sheath, and through which the optic nerve CSF could be drained out. To investigate whether lymphatic vessels are present in the optic nerve sheath, three lymphatic vessels-specific markers (LYVE-1, PDPN, FOXC2) were used to perform immunofluorescence staining on rat and human optic nerve sheaths and, followed by confocal microscopy imaging. The skin of rat and human were harvested as a positive control. In rats sampkles all three markers were observed in skin and optic nerve sheath, with a visible pattern of co-localization (Fig. 5 a). In human samples, the three markers were also detected in skin and optic nerve sheath, also with a pattern of co-localization (Fig. 5 b). Importantly, in both rat and humans samples, the positive areas in the optic nerve sheath exhibited both linear and tubular-like structures, characteristic of lymphatic vessels (Fig. 5 b). 5.The optic nerve CSF drains through the lymphatic vessels in the optic nerve sheath To investigate whether the CSF of the optic nerve is drained through lymphatic vessels in the optic nerve sheath, we injected ink into the sub-sheath space of the optic nerve in rats. Thirty minutes after injection, the optic nerve sheath was harvested and stained with lymphatic vessels-specific markers (LYVE-1, PDPN, FOXC2)(Fig. 6 a). Under light microscope, ink particles were identified in the lymphatic vessels-specific positive regions of the optic nerve sheath (Fig. 6 b). This indicates that the lymphatic vessels of the optic nerve sheath functioned to drain the optic nerve CSF. Discussion In this study,we found that in rats the optic nerve CSF does not flow back into the intracranial space, but, instead, drained into the dcLNs via the lymphatic vessels in the optic nerve sheath. This may provides important insights for better understanding the physiological turnover and drainage pathways of optic nerve CSF, as well as the pathology of SANS. The local circulation of optic nerve CSF consists of two parts: inflow and outflow. The pathway of CSF inflow is well-known. CSF (produced by the choroid plexus of the ventricles) flows through the third and fourth ventricles into the subarachnoid space, enters the optic canal from the optic chiasmal cystern and ultimately flows into the sub-sheath space of the optic nerve. There, the CSF exchanges substances with the optic nerve, maintaining its structural and functional stability[ 16 ]. However, the outflow of optic nerve CSF remains unclear. The traditional theory suggests that, optic nerve CSF flows back into the intracranial chiasmal cystern via the optic canal, and then mixes with the intracranial CSF; the mixture drains via the arachnoid granulations into the venous sinuses, ultimately returning to the bloodstream Whether optic nerve CSF undergoes a "bidirectional free flow" between the optic nerve and the chiasmal cystern via the optic canal has been inconclusive[ 10 , 17 , 18 ]. However, recent anatomic studies have challenged this traditional theory. In monkeys and humans, Killer et al. observed varying degrees of adhesion within the optic nerve canal, leading to the loss of free communication[ 10 ]. Hayreh and colleagues found that capillary trabecular networks extended from the periosteum into the optic nerve canal in the rabit[ 18 ]. Additionally, by electron microscopy, Killer et al. discovered fine anatomical structures such as trabeculae, septa, and pillars in the sub-sheath space of the optic nerve of humans[ 19 ]. The above anatomical findings indicate that the space within the optic canal is extremely narrow, with a clear separation between optic nerve CSF and intracranial CSF. This structural evidence contradicts the traditional view that CSF freely flows between these two spaces, thus challenging the traditional belief that optic nerve CSF drains into the intracranial space[ 10 , 12 , 20 ]. More recently, Liugan et al., using epoxy resin embedding technology, found a dense trabecular network inside the optic canal in human cadavers, as well as fusion between the optic canal and the arachnoid membrane, with some fibers of the optic nerve sheath attached to the periosteum of the sphenoid bone[ 21 ]. These new anatomical findings implies that, the optic nerve CSF is unlikely to "freely reflux" into the intracranial space via the optic canal[ 13 ]. Clinically, Killer et al. studied patients with idiopathic intracranial hypertension (IIH), and found a concentration gradient of beta-protein (a precursor of prostaglandin D-synthase) between lumbar and optic nerve CSF[ 20 ]. Subsequently, by using computed cisternography with the help of a contrast agent administered into patients with optic disc edema and normal-tension glaucoma, they found that CSF tracers were predominantly distributed in the intracranial part of the optic chiasm[ 14 , 20 ]. Based on these observations, they suggested that the flow of CSF between the intracranial space and the sub-sheath space of the optic nerve may be neither continuous nor bidirectional[ 10 ]. In this study, first, we measured the PSSON and ICP in rabbits to probe whether there is a pressure difference between the optic nerve CSF and the intracranial CSF. According to the principles of fluid dynamics, if optic nerve CSF and intracranial CSF are freely communicated, PSSON and ICP should be equal. However, exisiting studies on PSSON have been scarce, and the method for measuring PSSON has hardly been established because of the deep location of the optic nerve and obstruction of the eyeball. The earliest measurement of PSSON in human cadavers was reported by Liu et al. in 1993 with a handheld electronic digital manometer (Stryker). They found a considerable individual variation in PSSON, making it impossible to reach reliable conclusions[ 22 ]. In 1995, Liu et al. measured PSSON again in patients undergoing eye enucleation by a medial orbital approach, and their results showed a wide variation in PSSON, ranging from 4–14 mmHg (42–180 mmH 2 O)[ 17 ]. Their measurements, thus, did not indicate a clear relationship between ICP and PSSON. This may be attributable, at least partly, to the large individual variation, which was linked to the measurement instrument used. The manometer used in their study was directly connected to an 18 G needle via a rigid port. This configuration making it difficult for the manometer to access anatomically deep and inconvenient locations such as the sub-sheath space of the optic nerve in the orbit, which is blocked by the eyeball. Furthermore, the manometer is only precise to 1 mmHg (i.e.,13.6 mmH 2 O), which is insufficient for detecting subtle change of fluid pressures (i.e., mmH 2 O level). To address these limitations, Wang et al. implanted pressure-sensing probes into the left ventricle, lumbar cistern, optic nerve subarachnoid space, and anterior chamber in eight normal dogs in 2016 to examine the interdependence of ICP and intraocular pressure (IOP) and how it affects optic nerve pressures. Their results showed that all examined pressures were different (ICP > lumbar cistern pressure (LCP) > ONSP) at baseline. As ICP was lowered during CSF shunting, once ICP fell below a critical breakpoint, ICP and IOP became uncoupled, and the trans-lamina cribrosa gradient (TLPG) changed as ICP declined, resulting in an "ICP-IOP independent zone"[ 23 ]. To overcome the aforementioned limitations, we customized a fluid pressure tester. The instrumen is equipped with a small (25 G) needle, a soft pressure-transmission silicon tube, and a precision electronic pressure sensor (precise to 0.01 mmH 2 O). This design enables accurate and precise measurement of small fluid pressures in small or narrow spaces such as the sub-sheath space of the optic nerve (Fig. 1 a and 1 b). Calibration with a standard pressure gauge (Y055A, ZHT, XI’AN, China) confirmed excellent linearity and accuracy (Fig. 1 c). With this new tool, we measured both ICP and PSSON in normal rabbits, and found a PSSON range of 6.91-15.00 mmH 2 O (12.88 ± 2.94) and an ICP range of 15.8–26.4 mmH 2 O (20.88 ± 3.75)(Fig. 2 b). Subsequently, we made a rabbit model of intracranial hypertension (simulating the elevated intracranial pressure of astronauts under microgravity). In this model, PSSON was also significantly lower than ICP. These findings imply that, if optic nerve CSF attempts to flow back into the intracranial space, it needs to overcome the pressure difference between the two spaces. Evidently, this contradicts the traditional theory (i.e., optic nerve CSF refluxes into the intracranial space). To investigate whether the optic nerve CSF drains into the intracranial space and identify possibly alternative drainage routes, we injected fluorescence tracers into the sub-sheath space of the optic nerve of rats. Fluorescence spectrometry detected extremely low levels of the tracer in the optic chiasm (Fig. 3 b), which was likely due to background fluorescence or noise (e.g., laboratory light sources). Even if reflux occurred, the large intracranial volume could potentially dilute the tracer below the detection limit. This limitation highlights a significant challenge in the experimental design. Future studies should incorporate intracranial injection control groups and employ more sensitive tracing techniques. This is the first time to find that optic nerve CSF does not reflux into the intracranial space, implying the existence of alternative, previously undiscovered, outflow pathways. Recent discoveries of meningeal lymphatic vessels, which drain CSF and play a crucial role in CSF turnover, metabolic waste removal, and the maintenance of normal brain structure and function, are highly relevant in this context[ 15 , 24 – 26 ].We speculated that the optic nerve sheath, as an extension of the meninges into the orbit, should also have lymphatic vessels. Lymphatic vessels were once believed to be absent in the optic nerve and its surrounding sheath[ 27 ]. However, in 1985, Shen et al. first discovered a new CSF drainage pathway involving the subarachnoid space, sclera, and orbital connective tissue in rabbits and hamsters. But, they could not confirm whether CSF was drained out via the orbital lymphatic vessels[ 28 ]. Though the presence of lymphatic vessels was still in uncertainty, in 1999 "lymphatic-capillary-like structures" were first found and described in the human optic nerve sheath by transmission electron microscopy[ 19 ]. In 2004, Ludemann et al. injected tracers into the sub-sheath space of optic nerve in cats. They found tracers exited through fenestrated openings in the distal optic nerve sheath and were subsequently found in conjunctival lymphatic vessels. But they still did not confirm the presence of lymphatic vessels in the orbit neither in the optic nerve sheath[ 29 ]. In 2007, Gausas et al. found "vessels featuring the characteristics of lymphatics within the human optic nerve dura mater were detected by expression of the lymphatic-specific molecular marker D2-40".(The dura mater of the optic nerve in the original text refers to the optic nerve sheath studied in this project)[ 30 ]. With advancements in molecular biology, markers such as LYVE-1[ 31 – 33 ], PDPN[ 34 – 37 ], and FOXC2 [ 38 – 40 ] were found to be highly specific for identifying lymphatic vessels. In 2008, Masahide used immunohistochemistry with LYVE-1 to identify lymphatic vessels in the distal optic nerve sheath in mice, providing evidence of the presence of these vessels in the optic nerve region [ 41 ]. In our study, to reduce false posititvity, a combination of three antibodies (LYVE-1, PDPN, and FOXC2) were used to detect the presence of lymphatic vessels in both rat and human optic nerve sheaths (Fig. 5 ). To investigate that optic nerve CSF can be drained through the lymphatic vessels of the optic nerve sheath to the dcLNs, in this study the technique of CSF tracer was used and the high fluorescent intensity of tracer were found both in vivo and in vitro (Fig. 4 ). Furthermore, the method of immunofluorescence staining for lymphatic vessels in the optic nerve sheath and the method of CSF tracing were combined. It was found that the ink was in the lumen of the lymphatic vessels in the optic nerve sheath which was colocalized with three lymphatic vessels-specific markers (Fig. 6 ). Interestingly, in 2024 Song et al. also found a lymphatic drainage system in the anterior and posterior chambers of the eye. In their study, the posterior chamber of the eye drained to the dcLNs through the lymphatic vessels of the optic nerve sheath in mice[ 42 ]. In 2025 Jin et al. used fluorescent tracers in Prox1-GFP lymphatic reporter mice to map the pathway of CSF outflow through lymphatics to superficial cervical lymph nodes. They found that CSF entered initial lymphatics in the meninges at the skull base and continued through extracranial periorbital, olfactory, nasopharyngeal and hard palate lymphatics, and then through smooth muscle-covered superficial cervical lymphatics to submandibular lymph nodes[ 43 ]. Based on our findings and those of above researchers, we propose a new circulation for optic nerve CSF, that is, through the optic nerve canal, intracranial CSF flows into the optic nerve in the orbit, where it exchanges substances with the optic nerve. Then, the optic nerve CSF is drained out into the dcLNs via the lymphatic vessels in its sheath (Fig. 5 ). The importance of the lymphatic vessels in the optic nerve sheath and the discovered drainage pathway for optic nerve CSF is not yet fully understood. However, studies have shown that impaired drainage of CSF by meningeal lymphatic vessels —by a recently identified pathway to the dcLNs[ 15 , 24 – 26 ]—can lead to brain structural and functional damage, which is considered a pathophysiological mechanism in Alzheimer's disease, stroke, viral clearance and neuroinflammation[ 44 – 48 ]. Similarly, our findings may provide new biological and anatomical bases for the hypothesis that SANS may be related to the obstruction of lymphatic drainage in the optic nerve sheath [ 4 , 6 , 49 ] toward dcLNs, which could potentially contribute to the manifestation of SANS including increased ICP, elevated post-flight CSF pressure, optic disc edema, and optic nerve sheath expansion,in MRI [ 50 – 54 ]. Based on these insights, more targeted countermeasures could be developed to facilitate optic nerve CSF drainage through optic nerve sheath lymphatic pathways, complementing current strategies like lower-body negative pressure (LBNP)[ 55 – 58 ], nutritional supplementation[ 59 ], and centrifugation[ 60 ]. Notably, recent work by Jin et al.(2025)[ 43 ]reported that non-invasive mechanical stimulation of the superficial cervical lymphatics significantly increased CSF outflow in aged mice, offering a promising direction to enhance lymphatic drainage via cervical routes in astronauts. The limitations of this study include afert entering into the lymphatic vessels of the optic nerve sheath, the rout through which the CSF in the sheath is drained into the dcLNs remains unclear. In this study, we found that, ninety minutes after injection of Qdot655 into the sub-sheath space of the right optic nerve (Fig. 4 d), high-intensity fluorescence was detected in both the left and right dcLNs, and the fluorescence intensity in the injected side is significantly higher than in the contralateral side (Fig. 4 b). Whether this drainage occurs via meningeal lymphatic vessels, orbital periosteum lymphatic vessels or olfactory lymphatic vessels needs to be determined. Additionally, due to the limitations of the subjects, we were unable to investigate the outflow pathways of optic nerve CSF in humans. At present, we have not pathological models for SANS to validate the role of this pathway in the optic nerve. In summary, our study examined the circulation of optic nerve CSF and showed that it does not reflux into the intracranial space but instead, drains through the lymphatic vessels of the optic nerve sheath into the dcLNs. These findings not only help better understand the pathophysiology of SANS, and also provide a potential pharmacological or physical intervention strategy to protect astronauts’ vision during long-duration space missions by enhancing lymphatic drainage. Method Experimental model and study participants details Animals Forty Sprague–Dawley rats (6-9 weeks, 1.9 ± 0.1 kg, male:female = 1:1) were purchased from Shanghai Zhili Biological Company and housed in temperature- and humidity-controlled rooms maintained on a 12 h/12 h light/dark cycle (lights on at 7:00). All animals were kept under identical housing conditions. All procedures complied with regulations of the Institutional Animal Care and Use Committee at Shanghai General Hospital. Forty New Zealand White rabbits (50% male, 50% female; mean body weight: 1.9 ± 0.1 kg) were included in the experiment. Human samples Human skin samples were donated by patients undergoing blepharoplasty. Human optic nerve sheaths were donated by patients who experienced traumatic eyeball rupture and subsequently underwent enucleation performed at the National Clinical Research Center for Eye Diseases (Shanghai, China). All samples were collected under written informed consents from the patients. Ethical approval This study was reviewed and approved by the Medical Ethical Committee of the Shanghai General Hospital (2020-N–145). Each patient provided a written informed consent to be included in the study. The study was performed in accordance with the Declaration of Helsinki and its later amendments. Tissue preparation Tissue samples were fixed in 4% paraformaldehyde for 1 h at 20°C, rinsed in 0.1 M phosphate buffer (PBS, pH 7.4, 4°C, overnight) and transferred into PO 4 containing 15% sucrose (4°C, 24 h). Subsequently, they were embedded in tissue embedding medium (G6059, ServiceBio, Wuhan, Hubei, China), frozen at -80°C using liquid nitrogen-cooled iso-pentane, and stored at −20°C until further processing. Development and calibration of a pressure tester for measuring the pressure of the subarachnoid space of the optic nerve (PSSON) A pressure tester was designed based on Pascal’s principle. It consists of a testing needle connected via a silicon rubber tube (Pharmed BPT, Saint-Bobain, Akron, OH, USA), a mini-diaphragm pump (KLP, Kamoer Tech, Shanghai, China), a solenoid one-way valve (Vasco, Florham Park, NJ, USA), and a differential-capacitance pressure transmitter (measurement range: 0–80 mmH 2 O, accuracy: ±0.25%; Rosemount 3051C; Emerson, St. Louis, MO, USA). The one-way valve serves to prevent backflow of the saline. For testing, the needle is uninstalled. The valve opens and the pump draws sterile saline into the silicon tube till all air in the tube is displaced for 3 min. Then, the needle is capped and, and the saline is pumped again to displace the air in the needle for 2 s. Thus, the needle, silicon tube and the electric sensor are connected by a static liquid, allowing measurement of the hydraulic pressure at the tip.The pressure signal read by the electric sensor is displayed on an equipped LCD monitor, and also transmitted to a computer for storage and analysis. The device was calibrated with a pneumatic pressure gauge (Y055A, ZHT Instrument, Xian, Shaanxi, China). Measurement of PSSON in rabbits PSSON of 12 New Zealand white rabbits (1.8-2.0 kg, male:female = 1:1) were measured with the aforementioned pressure tester. The animal was anesthetized by injection of 3% sodium pentobarbital (1 ml/kg) into the marginal ear vein in conjunction with topical ophthalmic anesthesia and local injection of lidocaine into the skin at the lateral canthus. The lateral canthus was incised, and the globe was exposed with an eyelid speculum. The conjunctiva was incised from the 6 o’clock to 12 o’clock. Conjunctival peritomy was performed. The medial sclera and medial rectus muscle were exposed. The medial rectus muscle was isolated with a strabismus hook and then dissected with Westcott scissors. The muscle and conjunctiva were reflected nasally. The globe was rotated laterally with forceps. Small malleable retractors or cotton-tipped applicators were inserted along the medial globe and the orbital fat was retracted from the optic nerve. Then, the optic nerve sheath was exposed. After removing the microprobe, a 25 G needle was connected to the silicon tube of the pressure tester and the needle tip was placed adjacent to the optic nerve sheath and turn on the tester. The pressure outside the optic nerve sheath (P1) was measured with the tester. After 30 s, the needle tip was advanced to penetrate the optic nerve sheath and in the sub sheath space, and another pressure (P2) was measured. The difference (P2-P1) was calculated and recorded as the PSSON. Three measurements were made with 1 min interval between two consecutive ones. Measurement of intracranial pressure (ICP) in rabbits The rabbit was anesthetized as described above. The skin on the skull was incised to expose the cranium. A hole (diameter: 2 mm) was drilled on the cranium with an electric microdrill (RWD Life Science, Shenzhen, Guangdong, China) and sealed with a rubber plug manually made from a surgial glove. A 25 G needle (pre-connected to the silicon tube of the pressure tester) was placed on the surface of the hole. The pressure outside the cranial space (P1) was measured. After 30 s, the needle tip was advaned to penetrate the rubber plug (approximately 2 mm under the cranial bone), and another pressure (P2) was measured. The difference (P2-P1) represented the ICP. Three measurements were made with 1 min interval between two consecutive ones. Animal model of intracranial hypertension (ICH) The rabbit was anesthetized as described above, and the skin on the cranium was incised to expose the anterior fontanel. Two holes (diameter: 2 mm) were drilled with the electric microdrill and each sealed with a rubber plug. A bag of 0.9% saline solution was placed 30 cm above the head of the rabit, and continuously infused via a ruber plug into the cranial space, thereby creaing a model of intracranial hypertension. After infusion for 40 min, the ICP and PSSON were measured as described before. Injection of tracer for optic nerve CSF and intracranial CSF Rats were generally anesthetized by intraperitoneal injection of 10% chloral hydrate. Local anesthesia was performed with proparacaine hydrochloride eye drops for both eyes and lidocaine for the the skin at the lateral canthus. The lateral canthus was incised, and the conjunctiva was cut along the 6 o'clock to 12 o'clock direction. The muscles were dissected, and the conjunctiva was lifted with hemostatic clamp to expose the optic nerve sheath. Fluorescent tracer (Alexa Fluor 488, 2µL; dilution 1:100, Jackson ImmunoResearch, West Grove, PA, USA) was injected into the sub-sheath space of the optic nerve with a 10 µL microinjector (Outer diameter of the needle tip: 0.7 mm; Needle length: 51 mm, 34 G, Hamilton, Switzerland).In this study, we fixed the injection under the optic nerve sheath in the right eye. In vitro quantification of the tracer Alexa Fluor 488 To determine whether the tracer has refluxed into the intracranial compartment, 2 µL of Alexa Fluor 488 (dilution 1:100, Jackson Immuno Research, West Grove,PA,USA) was injected into the sub-sheath space of the optic nerve in rats by a microinjector (10 µL, Outer diameter of the needle tip: 0.7 mm; Needle length: 51 mm, 34 G, Hamilton,Switzerland). At 10, 20, and 30 min after injection, CSF samples were collected from the optic nerve and the optic chiasm (10 µL from each), respectively, and immediately measured for fluorescence spectrum (excitation: 495nm, emission: 520 nm; Hitachi F-700, Britain). To detect whether the tracer flows into the dcLNs, 2 µL of Alexa Fluor 488 (dilution 1:100, Jackson Immuno Research, West Grove,PA,USA) was injected into the sub-sheath space of the optic nerve in rats. At 30 min after injection,a CSF sample was collected from the optic nerve,and the ipsilateral dcLN was collected and homogenized to a slurry sample.Both samples were immediately analyzed for fluorescence intensity using the aforementioned spectrofluorometer (excitation: 495 nm,emission: 520 nm). In vivo fluorescent imaging for Qdot655 To detect whether Qdot655 flows into the dcLNs, 2 µL of Qdot655 (2 µL, dilution 1:100, Thermofisher, USA) was injected into the sub-sheath space of the optic nerve by a microinjector (10 µL, Outer diameter of the needle tip: 0.7 mm; Needle length: 51 mm, 34 G, Hamilton, Switzerland). At 90 min after injection, bilateral dcLNs were harvested and immediately measured with the in vivo imaging system for fluorescence emission (excitation: 405 nm, emission: 660 nm). Immunofluorescence Staining Human optic nerve sheath samples were obtained from patients with TON (tramatic optic neuropathy) who underwent optic nerve sheath fenestration, and human skin (positive control) samples were from the eyelid skin of patients who underwent blepharoplasty. Samples were fixed with 4% paraformaldehyde for 24 hours at room temperature and dehydrated in 10%, 20%, and 30% sucrose solutions for 24 hours at each concentration. The samples were then cut into 10 µm frozen tissue sections using a cryostat and stored at room temperature overnight. Tissue sections were rinsed three times with PBS (3-5 minutes each), followed by blocking with 2% BSA at room temperature or 37°C for 1 hour. Primary antibodies (anti-LYVE-1, dilution 1:200, Novus, Cat#: NB100-725; anti-FoxC2, dilution 1:200, R&D Systems, Cat#: MAB5044; and anti-PDPN, dilution 1:200, Novus, Cat#: NB600-1013) were applied, and the samples were incubated overnight at 4°C. After rinsing with PBS three times (3-5 minutes each), secondary antibodies (Alexa Fluor 488, dilution 1:500, Jackson,Cat#: 711-546-152; Alexa Fluor 594, dilution 1:500, Jackson, Cat#: 711-586-150; and Alexa Fluor 647, dilution 1:500, Jackson,Cat#: 705-606-147) were applied at room temperature or 37°C for 1 hour in the dark. After rinsing again with PBS (3 times, 3-5 minutes each), DAPI staining was performed for 10 minutes, followed by a final rinse with PBS (3 times, 3-5 minutes). The samples were mounted with an anti-fade mounting medium and observed under a confocal microscope (Leica). CSF Tracing technique Combined with Immunofluorescence Staining for Lymphatic Vessels A 10 µL Hamilton microinjector was used to inject 1 µL of ink into the sub-sheath space of the optic nerve in rats. Thirty minutes later, the optic nerve and its sheath were harvested and fixed in 4% paraformaldehyde for 24 hours. The tissues were dehydrated at room temperature in 10%, 20%, and 30% sucrose solutions for 24 hours each. The tissues were then cut into 10 µm continuous frozen sections using a cryostat and stored at room temperature overnight (the first section was left untreated, while subsequent sections underwent immunofluorescence staining). Tissue sections were rinsed three times with PBS (3-5 minutes each) and blocked with 2% BSA at room temperature or 37°C for 1 hour. Primary antibodies (anti-LYVE-1, dilution 1:200, Novus; anti-FoxC2, dilution 1:200, R&D Systems; and anti-PDPN, dilution 1:200, Novus) were applied and incubated overnight at 4°C. After rinsing with PBS (3 times, 3-5 minutes each), secondary antibodies (Alexa Fluor 488, dilution 1:500, Jackson; Alexa Fluor 594, dilution 1:500, Jackson; and Alexa Fluor 647, dilution 1:500, Jackson) were applied for 1 hour at room temperature or 37°C in the dark. Following rinsing with PBS (3 times, 3-5 minutes each), DAPI staining was performed for 10 minutes. After a final rinse with PBS (3 times, 3-5 minutes each), the samples were mounted with anti-fade mounting medium and observed under a confocal microscope (Leica SP8). The lymphatic vessel structures in the optic nerve sheath were compared with the first section to observe the presence of ink in the lymphatic structures. Quantification and statistical analysis Statistical significancy was evaluated by t-test, *p.value < 0.05; **p.value < 0.01; ***p.value < 0.001; ****p.value < 0.0001. Further details regarding number of experimental repeats or number of analyzed cells and graph description, are reported in figure legends. Declarations Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hui Chen ( [email protected] ). Materials availability This study did not generate new unique reagents. Data and code availability All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Antibodies The following primary antibodies were used to immunostain all tissues: anti-LYVE-1 (1:200, Novus, NB100-725), anti-FoxC2 (1:200, R&D Systems, MAB5044), and anti-PDPN (1:200, Novus, NB600-1013). Primary antibodies were detected using Alexa Fluor 488 (1:500, Jackson, 711-546-152), 594 (1:500, Jackson, 711-586-150), and 647 (1:500, Jackson,705-606-147) secondary antibody conjugates. Acknowledgments We thank the participants of the study. This work was supported by the Open Project of the National Facility for Translational Medicine (Shanghai)(TMSK02021-103), the Fundamental Research Funds for the Central Universities (No. YG2023ZD17), Department of Science and Technology of Sichuan Province, China (2020YFSY0044) Space Medical Experiment Project of China Manned Space Program (HYZHXMH01003) and National Natural Science Foundation of China (81230029) Author contributions H.C. conceptualized and designed the study, and developed the experimental protocols with Y.X. H.C., H.W., N.L.W., and K.D. designed and fabricated the PSSON pressure detector. Y.X., L.C., M.Y., Y.X.L., L.Y., Q.Y.Y., Y.W.H., W.J.C., X.Y.W., and L.Y.X. performed the experiments and analyzed the data. Y.X., W.J.C., Y.W.H., and M.Y. conducted the statistical analysis. Y.X., W.J.C., and Y.W.H. produced and annotated the images, and wrote the original draft. W.J.C.,Y.C., Y.X., K.D., X.X.R., L.C., N.L.W., and H.C. reviewed and edited the manuscript. Competing interests The authors declare no competing interests. Additional information Author information These authors contributed equally: Yang Xu, Wenjing Chen Authors and Affiliations Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Yang Xu, Min Yan, Lu Cheng, Hui Chen National Clinical Research Center for Eye Diseases, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Lu Cheng, Hui Chen Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai, China. Lu Cheng, Hui Chen Department of Ophthalmology, The Third People's Hospital of Zhangjiagang, Zhangjiagang, China. Yang Xu Eye School of Chengdu University of TCM, Chengdu, China. Yang Xu, Min Yan, Hui Chen Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China. Ningli Wang University of Electronic Science and Technology of China, Chengdu, China . Wenjing Chen, Yuwei Hu, Hui Chen University of Shanghai for Science and Technology, Shanghai, China. Yuanxi Lin Shanxi Eye Hospital, Taiyuan, Shanxi, China. Xiaoxia Ren Ophthalmology Department, Eastern Hospital, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu, China. Huan Wang Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China. Yuwei Hu Center for Big Data & Analytics, Shenzhen People's Hospital. Shenzhen, China. Xiaoyun Wu Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Sichuan Provincial Laboratory of Orthopedic Implant Device R&D and Application Technology Engineering, Luzhou, China Ke Duan School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, China Wenjing Chen Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China Yuan Xie Department of Ophthalmology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi’an, Shaanxi, China Ying Cheng, *:Co-c orresponden ts and requests for materials should be addressed to Hui Chen, MD, Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Hai Ning Road, Shanghai 200080, P.R. China; E-mail: [email protected] Ning-Li Wang, MD, Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology & Visual Sciences, Beijing 100730, P.R. China; E-mail: [email protected] Lu Cheng, MD, Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Hai Ning Road, Shanghai 200080, P.R. China; E-mail: [email protected] Ke Duan, PhD, Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Sichuan Provincial Laboratory of Orthopedic Implant Device R&D and Application Technology Engineering, Luzhou Sichuan, 646000, P. R. China; E-mail: [email protected] Xiaoxia Ren, MS, Shanxi Eye Hospital, Taiyuan, Shanxi 030002, P.R. China; E-mail: [email protected] Reprints and permissions information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Petersen, L.G., et al., Lower body negative pressure to safely reduce intracranial pressure . The Journal of Physiology, 2018. 597(1): p. 237–248. Workshop Program, in 2022 IEEE International Workshop on Sport, Technology and Research (STAR) . 2022, IEEE. p. 1–6. Sater, S.H., et al., MRI-based quantification of posterior ocular globe flattening during 60 days of strict 6° head-down tilt bed rest with and without daily centrifugation. Journal of applied physiology (Bethesda, Md.: 1985), 2022. 133(6): p. 1349–1355. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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10:59:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114522,"visible":true,"origin":"","legend":"\u003cp\u003ePrototype, design diagram, and calibration of the optic nerve sheath pressure measurement device.\u003c/p\u003e\n\u003cp\u003ea The scheme of the design of a pressure detector based on Pascal’s principle. b The pressure tester, silicon tube and its microprobe which can be replaced with a 25 G needle when measuring. c The calibration result against pneumatic pressure gauge.The pressure output by the pneumatic pressure gauge and the pressure from the tester (slope = 1.001 ± 0.001, p \u0026lt; 0.001; intercept = -0.030 ± 0.001, p = 0.967; R\u003csup\u003e2\u003c/sup\u003e = 1.0000).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/8b050dc49e549801ad3b448b.png"},{"id":87664572,"identity":"86de25a3-38e0-4ebc-9cbb-90c0d5a6ff1d","added_by":"auto","created_at":"2025-07-27 10:59:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of optic nerve sheath pressure and intracranial pressure in normal intracranial pressure rabbits and intracranial hypertension rabbit models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e The intracranial pressure (ICP) measurements in rabbits (upper image), and the pressure measurement in the sub-sheath space of the optic nerve (PSSON)(lower image). \u003cstrong\u003eb\u003c/strong\u003e Comparison of ICP and PSSON in normal rabbits (n = 12, paired t-test, ****p \u0026lt; 0.0001).\u003cstrong\u003e c\u003c/strong\u003e Comparison of ICP and PSSON in intracranial hypertension rabbit model (n = 10, paired t-test, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/42efc07a2fb50350560b343f.png"},{"id":87665629,"identity":"f205088f-e8e3-43b8-a907-8c3e414edb46","added_by":"auto","created_at":"2025-07-27 11:07:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the sub-sheath space of the optic nerve CSF and optic chiasm CSF tracer (Alexa Fluor 488) measurements in rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic of tracer Alexa Fluor 488 injection and measurement in the sub-sheath space of the optic nerve. \u003cstrong\u003eI\u003c/strong\u003e: Optic nerve CSF, \u003cstrong\u003eII\u003c/strong\u003e: Optic nerve sheath, \u003cstrong\u003eIII\u003c/strong\u003e: Optic chiasm CSF,\u003cstrong\u003e IV\u003c/strong\u003e: Deep cervical lymph nodes (dcLNs). Alexa Fluor 488 was injected into the sub-sheath space of the optic nerve in rats. CSF was collected at 10, 20, and 30 minutes after injection at the optic nerve (Region I of Fig. 3a) and optic chiasm cystern (Region III of Fig. 3a). \u003cstrong\u003eb\u003c/strong\u003e Comparison of fluorescence intensity between the optic nerve CSF (Region I of Fig. 3a) and optic chiasm CSF (Region III of Fig. 3a). Fluorescence intensity in optic nerve CSF decreased over time, while the fluorescence intensity in optic chiasm CSF was extremely low (n = 3, t-test, *p \u0026lt; 0.05, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/cc77ffef4568ad6b414115b1.png"},{"id":87666254,"identity":"a13525b3-5b63-4fea-8961-4064e2f59ce9","added_by":"auto","created_at":"2025-07-27 11:15:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":198246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInjection of tracers (Qdot655 and Alexa Fluor 488) into the sub-sheath space of the optic nerve, and comparison of intensity between the deep cervical lymph nodes (dcLNs) and optic nerve CSF in rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Anatomical image of the dcLNs in rats\u003cstrong\u003e. b\u003c/strong\u003e Ninety minutes after injection of 2 µL Qdot655 into the sub-sheath space of the right optic nerve (Fig. 4d), images were captured by using an in vivo imaging system. High-intensity fluorescence was detected in both the left and right dcLN, with the fluorescence intensity in the injected side significantly higher than in the contralateral side (\u003cstrong\u003ec\u003c/strong\u003e)(n = 3, t-test, **p＜0.01).\u003cstrong\u003e d\u003c/strong\u003e Schematic diagram of the sub-sheath space of the optic nerve (right) and dcLNs in rats (viewed from below). 30 minutes after injection, the optic nerve CSF was simultaneously collected, and the ipsilateral dcLNs was laso collected and crushed for fluorescence intensity analysis. \u003cstrong\u003ee\u003c/strong\u003eFluorescence intensity of Alexa Fluor 488 of the triturated dcLN tissue was measured by a fluorescence spectrometer, showing significantly higher fluorescence intensity compared to the optic nerve CSF (n = 6, t-test, ****p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e(R: right; L: left.dcLNs: deep cervical lymph nodes.ON-CSF: optic nerve CSF.)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/193ed2e6475348433975d5cf.png"},{"id":87664588,"identity":"fd3b4a50-fab9-449b-8147-fb3596682382","added_by":"auto","created_at":"2025-07-27 10:59:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":874229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLymphatic vessels in the optic nerve sheath of rats and humans (immunofluorescence staining)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eImmunofluorescence staining of lymphatic vessels in rat optic nerve sheath (rat skin as a positive control). All three lymphatic vessels-specific markers, LYVE-1, PDPN, and FOXC2, were positive, with co-localization regions observed.\u003cstrong\u003eb\u003c/strong\u003e Immunofluorescence staining of human skin and optic nerve sheath by using lymphatic vessels-specific markers (human skin as a positive control). LYVE-1, PDPN, and FoxC2, were all positive with co localized areas, exhibiting distinct linear and tubular-like structures.\u003c/p\u003e\n\u003cp\u003e(ONS: optic nerve sheath)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/19405357bb406351f57230c3.png"},{"id":87665630,"identity":"04be55c3-654c-4abe-acbf-70ce87b95452","added_by":"auto","created_at":"2025-07-27 11:07:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":330048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrainage of optic nerve CSF tracer (ink) through the lymphatic vessels in the optic nerve sheath of rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Using a 10 µL Hamilton microinjector, 1 µL of ink was injected into the sub-sheath space of the rat optic nerve. Thirty minutes after injection, frozen sections of the optic nerve and its sheath were harvested and stained by using lymphatic vessels-specific markers LYVE-1 (green), PDPN (red), and FOXC2 (white).The red dashed frame highlights a co-localized lumen-like structure by three lymphatic vessels-specific markers.\u003cstrong\u003eb \u003c/strong\u003eFrozen section without immunofluorescence staining was observed under a light microscope. Ink particles (blue arrows) were found within the co-localized lumen-like structure by three lymphatic vessels-specific markers in the sheath.(ON: optic nerve; ONS: optic nerve sheath)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/2a102018ec0fd19e71638651.png"},{"id":87664584,"identity":"42853fe1-3b20-4e3d-8948-107e1fe480e5","added_by":"auto","created_at":"2025-07-27 10:59:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":302606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of optic nerve CSF circulation and lymphatic vessels in optic nerve sheath.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e The intracranial CSF flows through the trabeculum and pillars of optic canal to the sub-sheath space of the optic nerve, where it exchanged with the optic nerve and then drained to the dcLNs through the lymphatic vessels of the optic nerve sheath. \u003cstrong\u003eb\u003c/strong\u003e An enlarged view of the optic nerve CSF circulation. The white reticular line in the optic canal represents the trabeculum and pillars, the green oval represents the optic nerve sheath lymphatic vessels and meningeal lymphatic vessels, and the white arrow indicates that the intracranial CSF enters the sub-sheath space of the optic nerve, where it exchanges nutrients and metabolic waste with the optic nerve. The green arrow indicates that the optic nerve CSF flows back to the dcLNs through the optic nerve sheath lymphatic vessels\u003cstrong\u003e. c\u003c/strong\u003e A stereoscopic view of the structure of the lymphatic vessels in the optic nerve sheath.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/b2f282ea49d42ebc558a3e30.png"},{"id":89773094,"identity":"f16c10a7-5c6d-4451-9554-6757c817ad74","added_by":"auto","created_at":"2025-08-24 18:46:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3913350,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7083215/v1/8992574d-4a07-48d4-9756-cc7a7704f472.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optic Nerve Cerebrospinal Fluid Drained-out via Its Sheath Lymphatic Vessels","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 2011, Mader et al. first reported the association between spaceflight and Spaceflight Associated Neuro-Ocular Syndrome (SANS), noting that astronauts exhibited optic nerve sheath expansion, optic disc edema, choroidal folds, globe flattening,and hypermetropic axial length changes following long-duration spaceflight[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Subsequent studies have shown that SANS may persist for years in some individuals[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe currently prevailing hypothesis suggests that these changes are caused by fluid redistribution in the microgravity environment[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], where alterations in gravity and hydrostatic pressure impact the transmural pressure in arteries, veins, and lymphatic vessels, leading to changes in local blood flow and lymphatic drainage. However, a critical question remains unresolved: Why do some post-flight changes, such as optic nerve cerebrospinal fluid (CSF) retention and optic nerve sheath expansion showed in magnetic resonance image (MRI), persist even long after astronauts return to Earth?\u003c/p\u003e\u003cp\u003eIn physiology, the CSF surrounding the optic nerve (to differentiate the with the intracranial CSF, it is named as optic nerve CSF) plays an important role in nutrient exchange, waste removal, and maintenance of optic nerve function.Recently, some studies suggested that, the optic disc edema observed in astronauts may be linked to the retention of the optic nerve CSF[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and this retention may be anatomically associated with impaired drainage of optic nerve CSF[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Some scholars argue that the elevation in the optic nerve sheath pressure (ONSP) due to microgravity-induced headward fluid shift is the primary hypothesized contributor to SANS[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These hypotheses highlight the urgent need to elucidate the circulation of optic nerve CSF.\u003c/p\u003e\u003cp\u003eTraditionally, it has been believed that intracranial CSF flows into and round the optic nerve through the optic canal, and subsequently drains back into the cranial cavity. Although the inflow pathway of optic nerve CSF is widely accepted, controversies remain as to whether it drains back into the intracranial space[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Killer et al. argued that it is unlikely for optic nerve CSF to flow back into the cranial cavity against the volume gradient, nor is it probable for bidirectional flow to occur within the optic canal[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Their perspectives are based on clinical evidences[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. But, if optic nerve CSF is not drained out through the optic canal, what alternative out-drainage pathway could it be?\u003c/p\u003e\u003cp\u003eIn past decades, a revolutionary theory proposed that lymphatic vessels within the meninge can drain intracranial CSF out into the deep cervical lymph nodes (dcLNs)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is well-known that the optic nerve is an anatomical extension of the central nervous system toward the orbit, surrounded by CSF and its sheath (an extension of the meninge). Thus, we hypothesize that, 1) lymphatic vessels may also exist in the optic nerve sheath, 2) optic nerve CSF may drain via these vessels.\u003c/p\u003e\u003cp\u003eTo verify these hypotheses, the present study focuses on the drainage pathway of optic nerve CSF.Our results demonstrate that, optic nerve CSF does not flow back into the intracranial space, but instead drains out into the dcLNs via lymphatic vessels in the optic nerve sheath. This discovery has significant implications for elucidating the drainage pathways of optic nerve CSF, and may be helpful for understanding the pathological nature of SANS.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1 The pressure in the sub-sheath space of optic nerve is lower than that in the intracranial space\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we designed and fabricated a pressure tester (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb) to measure the pressure in the sub-sheath space of the optic nerve (PSSON). The calibration of the pressure tester showed a strong linear correlation between the pressure output by the pneumatic pressure gauge and the tester, confirming its accuracy (slope\u0026thinsp;=\u0026thinsp;1.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; intercept = -0.030\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, p\u0026thinsp;=\u0026thinsp;0.967; R\u0026sup2; = 1.0000)(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eUsing the pressure tester, we measured the intracranial pressure (ICP) and the PSSON in rabbits (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). The PSSON was measured to be 12.88 \u0026plusmn; 2.94 mmH\u003csub\u003e2\u003c/sub\u003eO (n\u0026thinsp;=\u0026thinsp;12), while the ICP was 20.88 \u0026plusmn; 3.75 mmH\u003csub\u003e2\u003c/sub\u003eO. The PSSON was significantly lower than the ICP (n\u0026thinsp;=\u0026thinsp;12, paired t-test, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003eSubsequently, we established an intracranial hypertension model in rabbits by normal saline perfusion. In these rabbits, the PSSON was measured to be 273.7 \u0026plusmn; 6.00 mmH\u003csub\u003e2\u003c/sub\u003eO, and the ICP 301.4 \u0026plusmn; 1.4 mmH\u003csub\u003e2\u003c/sub\u003eO. Again the PSSON remained significantly lower than the ICP (n\u0026thinsp;=\u0026thinsp;10, paired t-test, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n\u003ch3\u003e2 The optic nerve CSF does not flow back into the intracranial space\u003c/h3\u003e\n\u003cp\u003eTo investigate whether optic nerve CSF flows back into the intracranial space, Alexa Fluor 488 was injected into the sub-sheath space of the right optic nerve in rats. It was found 10\u0026ndash;30 min after injection, the fluorescence intensity of Alexa Fluor 488 in the optic nerve CSF progressively decreased, indicating its drainage. Importantly, at each time point, the fluorescence intensity measured from the optic chiasm cystern CSF was markedly lower (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). These findings strongly suggest that, the optic nerve CSF did not flow back into the intracranial space.\u003c/p\u003e\n\u003ch3\u003e3. The optic nerve CSF flows to dcLNs\u003c/h3\u003e\n\u003cp\u003eTo study the outflow pathway of the optic nerve CSF, we injected the tracer Qdot655 into the sub-sheath space of the right optic nerve in rats. Ninety minutes later, strong fluorescence signals were detected in the right dcLN, with weaker fluorescence observed in the left dcLN (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eFurthermore, 30 minutes after the injection of Alexa Fluor 488 into the sub-sheath space of the right optic nerve (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed), fluorescence intensity in the dcLN was significantly higher than that in the optic nerve CSF (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee). Collectively, These findings supports the conclusion that optic nerve CSF flows to the dcLNs.\u003c/p\u003e\n\u003ch3\u003e4.Presence of lymphatic vessels in the optic nerve sheath\u003c/h3\u003e\n\u003cp\u003eIn past decades, a revolutionary theory was proposed, that lymphatic vessels within the meninge can drain intracranial CSF out into the dcLNs [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. It is well-known that the optic nerve is an anatomical extension of the central nervous system toward the orbit, surrounded by CSF and its sheath (an extension of the meninge). Thus, we hypothesize that, lymphatic vessels may also exist in the optic nerve sheath, and through which the optic nerve CSF could be drained out.\u003c/p\u003e\n\u003cp\u003eTo investigate whether lymphatic vessels are present in the optic nerve sheath, three lymphatic vessels-specific markers (LYVE-1, PDPN, FOXC2) were used to perform immunofluorescence staining on rat and human optic nerve sheaths and, followed by confocal microscopy imaging. The skin of rat and human were harvested as a positive control. In rats sampkles all three markers were observed in skin and optic nerve sheath, with a visible pattern of co-localization (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). In human samples, the three markers were also detected in skin and optic nerve sheath, also with a pattern of co-localization (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Importantly, in both rat and humans samples, the positive areas in the optic nerve sheath exhibited both linear and tubular-like structures, characteristic of lymphatic vessels (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\n\u003ch3\u003e5.The optic nerve CSF drains through the lymphatic vessels in the optic nerve sheath\u003c/h3\u003e\n\u003cp\u003eTo investigate whether the CSF of the optic nerve is drained through lymphatic vessels in the optic nerve sheath, we injected ink into the sub-sheath space of the optic nerve in rats. Thirty minutes after injection, the optic nerve sheath was harvested and stained with lymphatic vessels-specific markers (LYVE-1, PDPN, FOXC2)(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Under light microscope, ink particles were identified in the lymphatic vessels-specific positive regions of the optic nerve sheath (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). This indicates that the lymphatic vessels of the optic nerve sheath functioned to drain the optic nerve CSF.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study,we found that in rats the optic nerve CSF does not flow back into the intracranial space, but, instead, drained into the dcLNs via the lymphatic vessels in the optic nerve sheath. This may provides important insights for better understanding the physiological turnover and drainage pathways of optic nerve CSF, as well as the pathology of SANS.\u003c/p\u003e\u003cp\u003eThe local circulation of optic nerve CSF consists of two parts: inflow and outflow. The pathway of CSF inflow is well-known. CSF (produced by the choroid plexus of the ventricles) flows through the third and fourth ventricles into the subarachnoid space, enters the optic canal from the optic chiasmal cystern and ultimately flows into the sub-sheath space of the optic nerve. There, the CSF exchanges substances with the optic nerve, maintaining its structural and functional stability[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the outflow of optic nerve CSF remains unclear. The traditional theory suggests that, optic nerve CSF flows back into the intracranial chiasmal cystern via the optic canal, and then mixes with the intracranial CSF; the mixture drains via the arachnoid granulations into the venous sinuses, ultimately returning to the bloodstream Whether optic nerve CSF undergoes a \"bidirectional free flow\" between the optic nerve and the chiasmal cystern via the optic canal has been inconclusive[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, recent anatomic studies have challenged this traditional theory. In monkeys and humans, Killer et al. observed varying degrees of adhesion within the optic nerve canal, leading to the loss of free communication[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Hayreh and colleagues found that capillary trabecular networks extended from the periosteum into the optic nerve canal in the rabit[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, by electron microscopy, Killer et al. discovered fine anatomical structures such as trabeculae, septa, and pillars in the sub-sheath space of the optic nerve of humans[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The above anatomical findings indicate that the space within the optic canal is extremely narrow, with a clear separation between optic nerve CSF and intracranial CSF. This structural evidence contradicts the traditional view that CSF freely flows between these two spaces, thus challenging the traditional belief that optic nerve CSF drains into the intracranial space[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. More recently, Liugan et al., using epoxy resin embedding technology, found a dense trabecular network inside the optic canal in human cadavers, as well as fusion between the optic canal and the arachnoid membrane, with some fibers of the optic nerve sheath attached to the periosteum of the sphenoid bone[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These new anatomical findings implies that, the optic nerve CSF is unlikely to \"freely reflux\" into the intracranial space via the optic canal[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eClinically, Killer et al. studied patients with idiopathic intracranial hypertension (IIH), and found a concentration gradient of beta-protein (a precursor of prostaglandin D-synthase) between lumbar and optic nerve CSF[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Subsequently, by using computed cisternography with the help of a contrast agent administered into patients with optic disc edema and normal-tension glaucoma, they found that CSF tracers were predominantly distributed in the intracranial part of the optic chiasm[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on these observations, they suggested that the flow of CSF between the intracranial space and the sub-sheath space of the optic nerve may be neither continuous nor bidirectional[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, first, we measured the PSSON and ICP in rabbits to probe whether there is a pressure difference between the optic nerve CSF and the intracranial CSF. According to the principles of fluid dynamics, if optic nerve CSF and intracranial CSF are freely communicated, PSSON and ICP should be equal. However, exisiting studies on PSSON have been scarce, and the method for measuring PSSON has hardly been established because of the deep location of the optic nerve and obstruction of the eyeball. The earliest measurement of PSSON in human cadavers was reported by Liu et al. in 1993 with a handheld electronic digital manometer (Stryker). They found a considerable individual variation in PSSON, making it impossible to reach reliable conclusions[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In 1995, Liu et al. measured PSSON again in patients undergoing eye enucleation by a medial orbital approach, and their results showed a wide variation in PSSON, ranging from 4\u0026ndash;14 mmHg (42\u0026ndash;180 mmH\u003csub\u003e2\u003c/sub\u003eO)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Their measurements, thus, did not indicate a clear relationship between ICP and PSSON. This may be attributable, at least partly, to the large individual variation, which was linked to the measurement instrument used. The manometer used in their study was directly connected to an 18 G needle via a rigid port. This configuration making it difficult for the manometer to access anatomically deep and inconvenient locations such as the sub-sheath space of the optic nerve in the orbit, which is blocked by the eyeball. Furthermore, the manometer is only precise to 1 mmHg (i.e.,13.6 mmH\u003csub\u003e2\u003c/sub\u003eO), which is insufficient for detecting subtle change of fluid pressures (i.e., mmH\u003csub\u003e2\u003c/sub\u003eO level). To address these limitations, Wang et al. implanted pressure-sensing probes into the left ventricle, lumbar cistern, optic nerve subarachnoid space, and anterior chamber in eight normal dogs in 2016 to examine the interdependence of ICP and intraocular pressure (IOP) and how it affects optic nerve pressures. Their results showed that all examined pressures were different (ICP\u0026thinsp;\u0026gt;\u0026thinsp;lumbar cistern pressure (LCP)\u0026thinsp;\u0026gt;\u0026thinsp;ONSP) at baseline. As ICP was lowered during CSF shunting, once ICP fell below a critical breakpoint, ICP and IOP became uncoupled, and the trans-lamina cribrosa gradient (TLPG) changed as ICP declined, resulting in an \"ICP-IOP independent zone\"[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo overcome the aforementioned limitations, we customized a fluid pressure tester. The instrumen is equipped with a small (25 G) needle, a soft pressure-transmission silicon tube, and a precision electronic pressure sensor (precise to 0.01 mmH\u003csub\u003e2\u003c/sub\u003eO). This design enables accurate and precise measurement of small fluid pressures in small or narrow spaces such as the sub-sheath space of the optic nerve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Calibration with a standard pressure gauge (Y055A, ZHT, XI\u0026rsquo;AN, China) confirmed excellent linearity and accuracy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eWith this new tool, we measured both ICP and PSSON in normal rabbits, and found a PSSON range of 6.91-15.00 mmH\u003csub\u003e2\u003c/sub\u003eO (12.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94) and an ICP range of 15.8\u0026ndash;26.4 mmH\u003csub\u003e2\u003c/sub\u003eO (20.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Subsequently, we made a rabbit model of intracranial hypertension (simulating the elevated intracranial pressure of astronauts under microgravity). In this model, PSSON was also significantly lower than ICP. These findings imply that, if optic nerve CSF attempts to flow back into the intracranial space, it needs to overcome the pressure difference between the two spaces. Evidently, this contradicts the traditional theory (i.e., optic nerve CSF refluxes into the intracranial space).\u003c/p\u003e\u003cp\u003eTo investigate whether the optic nerve CSF drains into the intracranial space and identify possibly alternative drainage routes, we injected fluorescence tracers into the sub-sheath space of the optic nerve of rats. Fluorescence spectrometry detected extremely low levels of the tracer in the optic chiasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), which was likely due to background fluorescence or noise (e.g., laboratory light sources). Even if reflux occurred, the large intracranial volume could potentially dilute the tracer below the detection limit. This limitation highlights a significant challenge in the experimental design. Future studies should incorporate intracranial injection control groups and employ more sensitive tracing techniques. This is the first time to find that optic nerve CSF does not reflux into the intracranial space, implying the existence of alternative, previously undiscovered, outflow pathways.\u003c/p\u003e\u003cp\u003eRecent discoveries of meningeal lymphatic vessels, which drain CSF and play a crucial role in CSF turnover, metabolic waste removal, and the maintenance of normal brain structure and function, are highly relevant in this context[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].We speculated that the optic nerve sheath, as an extension of the meninges into the orbit, should also have lymphatic vessels.\u003c/p\u003e\u003cp\u003eLymphatic vessels were once believed to be absent in the optic nerve and its surrounding sheath[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, in 1985, Shen et al. first discovered a new CSF drainage pathway involving the subarachnoid space, sclera, and orbital connective tissue in rabbits and hamsters. But, they could not confirm whether CSF was drained out via the orbital lymphatic vessels[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Though the presence of lymphatic vessels was still in uncertainty, in 1999 \"lymphatic-capillary-like structures\" were first found and described in the human optic nerve sheath by transmission electron microscopy[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In 2004, Ludemann et al. injected tracers into the sub-sheath space of optic nerve in cats. They found tracers exited through fenestrated openings in the distal optic nerve sheath and were subsequently found in conjunctival lymphatic vessels. But they still did not confirm the presence of lymphatic vessels in the orbit neither in the optic nerve sheath[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In 2007, Gausas et al. found \"vessels featuring the characteristics of lymphatics within the human optic nerve dura mater were detected by expression of the lymphatic-specific molecular marker D2-40\".(The dura mater of the optic nerve in the original text refers to the optic nerve sheath studied in this project)[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. With advancements in molecular biology, markers such as LYVE-1[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], PDPN[\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and FOXC2 [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] were found to be highly specific for identifying lymphatic vessels. In 2008, Masahide used immunohistochemistry with LYVE-1 to identify lymphatic vessels in the distal optic nerve sheath in mice, providing evidence of the presence of these vessels in the optic nerve region [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In our study, to reduce false posititvity, a combination of three antibodies (LYVE-1, PDPN, and FOXC2) were used to detect the presence of lymphatic vessels in both rat and human optic nerve sheaths (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo investigate that optic nerve CSF can be drained through the lymphatic vessels of the optic nerve sheath to the dcLNs, in this study the technique of CSF tracer was used and the high fluorescent intensity of tracer were found both in vivo and in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, the method of immunofluorescence staining for lymphatic vessels in the optic nerve sheath and the method of CSF tracing were combined. It was found that the ink was in the lumen of the lymphatic vessels in the optic nerve sheath which was colocalized with three lymphatic vessels-specific markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, in 2024 Song et al. also found a lymphatic drainage system in the anterior and posterior chambers of the eye. In their study, the posterior chamber of the eye drained to the dcLNs through the lymphatic vessels of the optic nerve sheath in mice[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In 2025 Jin et al. used fluorescent tracers in Prox1-GFP lymphatic reporter mice to map the pathway of CSF outflow through lymphatics to superficial cervical lymph nodes. They found that CSF entered initial lymphatics in the meninges at the skull base and continued through extracranial periorbital, olfactory, nasopharyngeal and hard palate lymphatics, and then through smooth muscle-covered superficial cervical lymphatics to submandibular lymph nodes[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Based on our findings and those of above researchers, we propose a new circulation for optic nerve CSF, that is, through the optic nerve canal, intracranial CSF flows into the optic nerve in the orbit, where it exchanges substances with the optic nerve. Then, the optic nerve CSF is drained out into the dcLNs via the lymphatic vessels in its sheath (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe importance of the lymphatic vessels in the optic nerve sheath and the discovered drainage pathway for optic nerve CSF is not yet fully understood. However, studies have shown that impaired drainage of CSF by meningeal lymphatic vessels \u0026mdash;by a recently identified pathway to the dcLNs[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u0026mdash;can lead to brain structural and functional damage, which is considered a pathophysiological mechanism in Alzheimer's disease, stroke, viral clearance and neuroinflammation[\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSimilarly, our findings may provide new biological and anatomical bases for the hypothesis that SANS may be related to the obstruction of lymphatic drainage in the optic nerve sheath [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] toward dcLNs, which could potentially contribute to the manifestation of SANS including increased ICP, elevated post-flight CSF pressure, optic disc edema, and optic nerve sheath expansion,in MRI [\u003cspan additionalcitationids=\"CR51 CR52 CR53\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Based on these insights, more targeted countermeasures could be developed to facilitate optic nerve CSF drainage through optic nerve sheath lymphatic pathways, complementing current strategies like lower-body negative pressure (LBNP)[\u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], nutritional supplementation[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and centrifugation[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Notably, recent work by Jin et al.(2025)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]reported that non-invasive mechanical stimulation of the superficial cervical lymphatics significantly increased CSF outflow in aged mice, offering a promising direction to enhance lymphatic drainage via cervical routes in astronauts.\u003c/p\u003e\u003cp\u003eThe limitations of this study include afert entering into the lymphatic vessels of the optic nerve sheath, the rout through which the CSF in the sheath is drained into the dcLNs remains unclear. In this study, we found that, ninety minutes after injection of Qdot655 into the sub-sheath space of the right optic nerve (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), high-intensity fluorescence was detected in both the left and right dcLNs, and the fluorescence intensity in the injected side is significantly higher than in the contralateral side (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Whether this drainage occurs via meningeal lymphatic vessels, orbital periosteum lymphatic vessels or olfactory lymphatic vessels needs to be determined. Additionally, due to the limitations of the subjects, we were unable to investigate the outflow pathways of optic nerve CSF in humans. At present, we have not pathological models for SANS to validate the role of this pathway in the optic nerve.\u003c/p\u003e\u003cp\u003eIn summary, our study examined the circulation of optic nerve CSF and showed that it does not reflux into the intracranial space but instead, drains through the lymphatic vessels of the optic nerve sheath into the dcLNs. These findings not only help better understand the pathophysiology of SANS, and also provide a potential pharmacological or physical intervention strategy to protect astronauts\u0026rsquo; vision during long-duration space missions by enhancing lymphatic drainage.\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003e\u003cstrong\u003eExperimental model and study participants details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty Sprague\u0026ndash;Dawley rats (6-9 weeks, 1.9 \u0026plusmn; 0.1 kg, male:female = 1:1) were purchased from Shanghai Zhili Biological Company and housed in temperature- and humidity-controlled rooms maintained on a 12 h/12 h light/dark cycle (lights on at 7:00).\u0026nbsp;\u003cbr\u003eAll animals were kept under identical housing conditions. All procedures complied with regulations of the Institutional Animal Care and Use Committee at Shanghai General Hospital.\u003c/p\u003e\n\u003cp\u003eForty New Zealand White rabbits (50% male, 50% female; mean body weight: 1.9\u0026nbsp;\u0026plusmn;\u0026nbsp;0.1 kg) were included in the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esamples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman skin samples were donated by patients undergoing blepharoplasty. Human optic nerve sheaths were donated by patients who experienced traumatic eyeball rupture and subsequently underwent enucleation performed at the National Clinical Research Center for Eye Diseases (Shanghai, China). All samples were collected under written informed consents from the patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eapproval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Medical Ethical Committee of the Shanghai General Hospital (2020-N\u0026ndash;145). Each patient provided a written informed consent to be included in the study. The study was performed in accordance with the Declaration of Helsinki and its later amendments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epreparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples were fixed in 4% paraformaldehyde for 1 h at 20\u0026deg;C, rinsed in 0.1 M phosphate buffer (PBS, pH 7.4, 4\u0026deg;C, overnight) and transferred into PO\u003csub\u003e4\u003c/sub\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003econtaining\u0026nbsp;15%\u0026nbsp;sucrose\u0026nbsp;(4\u0026deg;C,\u0026nbsp;24\u0026nbsp;h).\u0026nbsp;Subsequently, they\u0026nbsp;were\u0026nbsp;embedded\u0026nbsp;in\u0026nbsp;tissue\u0026nbsp;embedding\u0026nbsp;medium\u0026nbsp;(G6059,\u0026nbsp;ServiceBio, Wuhan, Hubei, China),\u0026nbsp;frozen\u0026nbsp;at\u0026nbsp;-80\u0026deg;C\u0026nbsp;using\u0026nbsp;liquid\u0026nbsp;nitrogen-cooled\u0026nbsp;iso-pentane,\u0026nbsp;and\u0026nbsp;stored\u0026nbsp;at\u0026nbsp;\u0026minus;20\u0026deg;C\u0026nbsp;until\u0026nbsp;further\u0026nbsp;processing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment and calibration of a pressure tester for measuring the pressure of the subarachnoid space of the optic nerve (PSSON)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA pressure tester was designed based on Pascal\u0026rsquo;s principle. It consists of a testing needle connected via a silicon rubber tube (Pharmed BPT, Saint-Bobain, Akron, OH, USA), a mini-diaphragm pump (KLP, Kamoer Tech, Shanghai, China), a solenoid one-way valve (Vasco, Florham Park, NJ, USA), and a differential-capacitance pressure transmitter (measurement range: 0\u0026ndash;80 mmH\u003csub\u003e2\u003c/sub\u003eO, accuracy: \u0026plusmn;0.25%; Rosemount 3051C; Emerson, St. Louis, MO, USA). The one-way valve serves to prevent backflow of the saline. For testing, the needle is uninstalled. The valve opens and the pump draws sterile saline into the silicon tube till all air in the tube is displaced for 3 min. Then, the needle is capped and, and the saline is pumped again to displace the air in the needle for 2 s. Thus, the needle, silicon tube and the electric sensor are connected by a static liquid, allowing measurement of the hydraulic pressure at the tip.The pressure signal read by the electric sensor is displayed on an equipped LCD monitor, and also transmitted to a computer for storage and analysis.\u003c/p\u003e\n\u003cp\u003eThe device was calibrated with a pneumatic pressure gauge (Y055A, ZHT Instrument, Xian, Shaanxi, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of PSSON in rabbits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePSSON of 12 New Zealand white rabbits (1.8-2.0 kg, male:female = 1:1) were measured with the aforementioned pressure tester. The animal was anesthetized by injection of 3% sodium pentobarbital (1 ml/kg) into the marginal ear vein in conjunction with topical ophthalmic anesthesia and local injection of lidocaine into the skin at the lateral canthus.\u003c/p\u003e\n\u003cp\u003eThe lateral canthus was incised, and the globe was exposed with an eyelid speculum. The conjunctiva was incised from the 6 o\u0026rsquo;clock to 12 o\u0026rsquo;clock. Conjunctival peritomy was performed. The medial sclera and medial rectus muscle were exposed. The medial rectus muscle was isolated with a strabismus hook and then dissected with Westcott scissors. The muscle and conjunctiva were reflected nasally. The globe was rotated laterally with forceps. Small malleable retractors or cotton-tipped applicators were inserted along the medial globe and the orbital fat was retracted from the optic nerve. Then, the optic nerve sheath was exposed.\u003c/p\u003e\n\u003cp\u003eAfter removing the microprobe, a 25 G needle was connected to the silicon tube of the pressure tester and the needle tip was placed adjacent to the optic nerve sheath and turn on the tester. The pressure outside the optic nerve sheath (P1) was measured with the tester. After 30 s, the needle tip was advanced to penetrate the optic nerve sheath and in the sub sheath space, and another pressure (P2) was measured. The difference (P2-P1) was calculated and recorded as the PSSON. Three measurements were made with 1 min interval between two consecutive ones.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of intracranial pressure (ICP) in rabbits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rabbit was anesthetized as described above. The skin on the skull was incised to expose the cranium. A hole (diameter: 2 mm) was drilled on the cranium with an electric microdrill (RWD Life Science, Shenzhen, Guangdong, China) and sealed with a rubber plug manually made from a surgial glove. A 25 G needle (pre-connected to the silicon tube of the pressure tester) was placed on the surface of the hole. The pressure outside the cranial space (P1) was measured. After 30 s, the needle tip was advaned to penetrate the rubber plug (approximately 2 mm under the cranial bone), and another pressure (P2) was measured. The difference (P2-P1) represented the ICP. Three measurements were made with 1 min interval between two consecutive ones.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal model of intracranial hypertension (ICH)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rabbit was anesthetized as described above, and the skin on the cranium was incised to expose the anterior fontanel. Two holes (diameter: 2 mm) were drilled with the electric microdrill and each sealed with a rubber plug. A bag of 0.9% saline solution was placed 30 cm above the head of the rabit, and continuously infused via a ruber plug into the cranial space, thereby creaing a model of intracranial hypertension. After infusion for 40 min, the ICP and PSSON were measured as described before.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInjection of tracer for optic nerve CSF and intracranial CSF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were generally anesthetized by intraperitoneal injection of 10% chloral hydrate. Local anesthesia was performed with proparacaine hydrochloride eye drops for both eyes and lidocaine for the the skin at the lateral canthus. The lateral canthus was incised, and the conjunctiva was cut along the 6 o\u0026apos;clock to 12 o\u0026apos;clock direction. The muscles were dissected, and the conjunctiva was lifted with hemostatic clamp to expose the optic nerve sheath. Fluorescent tracer (Alexa Fluor 488, 2\u0026micro;L; dilution 1:100, Jackson ImmunoResearch, West Grove, PA, USA) was injected into the sub-sheath space of the optic nerve with a 10 \u0026micro;L microinjector (Outer diameter of the needle tip: 0.7 mm; Needle length: 51 mm, 34 G, Hamilton, Switzerland).In this study, we fixed the injection under the optic nerve sheath in the right eye.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro quantification of the tracer Alexa Fluor 488\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the tracer has refluxed into the intracranial compartment, 2 \u0026micro;L of Alexa Fluor 488 (dilution 1:100, Jackson Immuno Research, West Grove,PA,USA) was injected into the sub-sheath space of the optic nerve in rats by a microinjector (10 \u0026micro;L, Outer diameter of the needle tip: 0.7 mm; Needle length: 51 mm, 34 G, Hamilton,Switzerland). At 10, 20, and 30 min after injection, CSF samples were collected from the optic nerve and the optic chiasm (10 \u0026micro;L from each), respectively, and immediately measured for fluorescence spectrum (excitation: 495nm, emission: 520 nm; Hitachi F-700, Britain).\u003c/p\u003e\n\u003cp\u003eTo detect whether the tracer flows into the dcLNs, 2 \u0026micro;L of Alexa Fluor 488 (dilution 1:100, Jackson Immuno Research, West Grove,PA,USA) was injected into the sub-sheath space of the optic nerve in rats. At 30 min after injection,a CSF sample was collected from the optic nerve,and the ipsilateral dcLN was collected and homogenized to a slurry sample.Both samples were immediately analyzed for fluorescence intensity using the aforementioned spectrofluorometer (excitation: 495 nm,emission: 520 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo fluorescent imaging for Qdot655\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo detect whether Qdot655 flows into the dcLNs, 2 \u0026micro;L of Qdot655 (2 \u0026micro;L, dilution 1:100, Thermofisher, USA) was injected into the sub-sheath space of the optic nerve by a microinjector (10 \u0026micro;L, Outer diameter of the needle tip: 0.7 mm; Needle length: 51 mm, 34 G, Hamilton, Switzerland). At 90 min after injection, bilateral dcLNs were harvested and immediately measured with the in vivo imaging system for fluorescence emission (excitation: 405 nm, emission: 660 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman optic nerve sheath samples were obtained from patients with TON (tramatic optic neuropathy) who underwent optic nerve sheath fenestration, and human skin (positive control) samples were from the eyelid skin of patients who underwent blepharoplasty. Samples were fixed with 4% paraformaldehyde for 24 hours at room temperature and dehydrated in 10%, 20%, and 30% sucrose solutions for 24 hours at each concentration. The samples were then cut into 10 \u0026micro;m frozen tissue sections using a cryostat and stored at room temperature overnight. Tissue sections were rinsed three times with PBS (3-5 minutes each), followed by blocking with 2% BSA at room temperature or 37\u0026deg;C for 1 hour. Primary antibodies (anti-LYVE-1, dilution 1:200, Novus, Cat#: NB100-725; anti-FoxC2, dilution 1:200, R\u0026amp;D Systems, Cat#: MAB5044; and anti-PDPN, dilution 1:200, Novus, Cat#: NB600-1013) were applied, and the samples were incubated overnight at 4\u0026deg;C. After rinsing with PBS three times (3-5 minutes each), secondary antibodies (Alexa Fluor 488, dilution 1:500, Jackson,Cat#: 711-546-152; Alexa Fluor 594, dilution 1:500, Jackson, Cat#: 711-586-150; and Alexa Fluor 647, dilution 1:500, Jackson,Cat#: 705-606-147) were applied at room temperature or 37\u0026deg;C for 1 hour in the dark. After rinsing again with PBS (3 times, 3-5 minutes each), DAPI staining was performed for 10 minutes, followed by a final rinse with PBS (3 times, 3-5 minutes). The samples were mounted with an anti-fade mounting medium and observed under a confocal microscope (Leica).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCSF Tracing technique Combined with Immunofluorescence Staining for Lymphatic Vessels\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 10 \u0026micro;L Hamilton microinjector was used to inject 1 \u0026micro;L of ink into the sub-sheath space of the optic nerve in rats. Thirty minutes later, the optic nerve and its sheath were harvested and fixed in 4% paraformaldehyde for 24 hours. The tissues were dehydrated at room temperature in 10%, 20%, and 30% sucrose solutions for 24 hours each. The tissues were then cut into 10 \u0026micro;m continuous frozen sections using a cryostat and stored at room temperature overnight (the first section was left untreated, while subsequent sections underwent immunofluorescence staining). Tissue sections were rinsed three times with PBS (3-5 minutes each) and blocked with 2% BSA at room temperature or 37\u0026deg;C for 1 hour. Primary antibodies (anti-LYVE-1, dilution 1:200, Novus; anti-FoxC2, dilution 1:200, R\u0026amp;D Systems; and anti-PDPN, dilution 1:200, Novus) were applied and incubated overnight at 4\u0026deg;C. After rinsing with PBS (3 times, 3-5 minutes each), secondary antibodies (Alexa Fluor 488, dilution 1:500, Jackson; Alexa Fluor 594, dilution 1:500, Jackson; and Alexa Fluor 647, dilution 1:500, Jackson) were applied for 1 hour at room temperature or 37\u0026deg;C in the dark. Following rinsing with PBS (3 times, 3-5 minutes each), DAPI staining was performed for 10 minutes. After a final rinse with PBS (3 times, 3-5 minutes each), the samples were mounted with anti-fade mounting medium and observed under a confocal microscope (Leica SP8). The lymphatic vessel structures in the optic nerve sheath were compared with the first section to observe the presence of ink in the lymphatic structures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical significancy was evaluated by t-test, *p.value \u0026lt; 0.05; **p.value \u0026lt; 0.01; ***p.value \u0026lt; 0.001; ****p.value \u0026lt; 0.0001. Further details regarding number of experimental repeats or number of analyzed cells and graph description, are reported in figure legends.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eResource\u003c/strong\u003e\u003cstrong\u003eavailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLead contact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hui Chen ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not generate new unique reagents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following primary antibodies were used to immunostain all tissues: anti-LYVE-1 (1:200, Novus, NB100-725), anti-FoxC2 (1:200, R\u0026amp;D Systems, MAB5044), and anti-PDPN (1:200, Novus, NB600-1013). Primary antibodies were detected using Alexa Fluor 488 (1:500, Jackson, 711-546-152), 594 (1:500, Jackson, 711-586-150), and 647 (1:500, Jackson,705-606-147) secondary antibody conjugates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the participants of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Open Project of the National Facility for Translational Medicine (Shanghai)(TMSK02021-103), the Fundamental Research Funds for the Central Universities (No. YG2023ZD17), Department of Science and Technology of Sichuan Province, China (2020YFSY0044)\u0026nbsp;Space Medical Experiment Project of China Manned Space Program (HYZHXMH01003) and National Natural Science Foundation of China (81230029)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003econtributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.C. conceptualized and designed the study, and developed the experimental protocols with Y.X. H.C., H.W., N.L.W., and K.D. designed and fabricated the PSSON pressure detector. Y.X., L.C., M.Y., Y.X.L., L.Y., Q.Y.Y., Y.W.H., W.J.C., X.Y.W., and L.Y.X. performed the experiments and analyzed the data. Y.X., W.J.C., Y.W.H., and M.Y. conducted the statistical analysis. Y.X., W.J.C., and Y.W.H. produced and annotated the images, and wrote the original draft. W.J.C.,Y.C., Y.X., K.D., X.X.R., L.C., N.L.W., and H.C. reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional\u003c/strong\u003e\u003cstrong\u003einformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003einformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors contributed equally: Yang Xu, Wenjing Chen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eOphthalmology,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eShanghai\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eGeneral\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHospital,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eShanghai\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eJiao\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTong\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eUniversity\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSchool\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMedicine,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eShanghai,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang Xu, Min Yan, Lu Cheng, Hui Chen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNational Clinical Research Center for Eye Diseases, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLu Cheng, Hui Chen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShanghai\u003c/strong\u003e\u003cstrong\u003eKey\u003c/strong\u003e\u003cstrong\u003eLaboratory\u003c/strong\u003e\u003cstrong\u003eof\u003c/strong\u003e\u003cstrong\u003eOcular\u003c/strong\u003e\u003cstrong\u003eFundus\u003c/strong\u003e\u003cstrong\u003eDiseases,\u003c/strong\u003e\u003cstrong\u003eShanghai,\u003c/strong\u003e\u003cstrong\u003eChina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLu Cheng, Hui 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Ren\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOphthalmology\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDepartment,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEastern\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHospital,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSichuan\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcademy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMedical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSciences\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026amp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSichuan\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eProvincial\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePeople\u0026apos;s\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHospital,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChengdu,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuan Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitute\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBlood\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTransfusion,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChinese\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcademy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMedical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSciences\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePeking\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eUnion\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMedical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCollege,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChengdu,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuwei Hu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCenter\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efor\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBig\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026amp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAnalytics,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eShenzhen\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePeople\u0026apos;s\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHospital.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eShenzhen,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaoyun Wu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Sichuan Provincial Laboratory of Orthopedic Implant Device R\u0026amp;D and Application Technology Engineering, Luzhou, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKe Duan\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchool of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenjing Chen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBeijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuan Xie\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Ophthalmology, The First Affiliated Hospital of Xi\u0026apos;an Jiaotong University, Xi\u0026rsquo;an, Shaanxi, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYing Cheng,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*:Co-c\u003c/strong\u003e\u003cstrong\u003eorresponden\u003c/strong\u003e\u003cstrong\u003ets\u003c/strong\u003e and requests for materials should be addressed to\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHui Chen, MD, Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Hai Ning Road, Shanghai 200080, P.R. China; E-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eNing-Li Wang, MD, Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology \u0026amp; Visual Sciences, Beijing 100730, P.R. China; E-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eLu Cheng, MD, Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Hai Ning Road, Shanghai 200080, P.R. China; E-mail:[email protected]\u003c/p\u003e\n\u003cp\u003eKe Duan, PhD, Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Sichuan Provincial Laboratory of Orthopedic Implant Device R\u0026amp;D and Application Technology Engineering, Luzhou Sichuan, 646000, P. R. China; E-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eXiaoxia Ren, MS, Shanxi Eye Hospital, Taiyuan, Shanxi 030002, P.R. China; E-mail: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions information\u003c/strong\u003e is available at http://www.nature.com/reprints\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note\u0026nbsp;\u003c/strong\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u0026nbsp;\u003c/strong\u003eThis article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMader, T.H., et al., \u003cem\u003eOptic disc edema, globe flattening, choroidal folds, and hyperopic shifts observed in astronauts after long-duration space flight\u003c/em\u003e. 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The Journal of Physiology, 2018. 597(1): p. 237\u0026ndash;248.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWorkshop Program, in \u003cem\u003e2022 IEEE International Workshop on Sport, Technology and Research (STAR)\u003c/em\u003e. 2022, IEEE. p. 1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSater, S.H., et al., \u003cem\u003eMRI-based quantification of posterior ocular globe flattening during 60 days of strict 6\u0026deg; head-down tilt bed rest with and without daily centrifugation.\u003c/em\u003e Journal of applied physiology (Bethesda, Md.: 1985), 2022. 133(6): p. 1349\u0026ndash;1355.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"optic nerve CSF, sub-sheath space of the optic nerve, lymphatic vessels, optic nerve sheath, spaceflight associated neuro-ocular syndrome (SANS), intracranial and intraocular pressure difference","lastPublishedDoi":"10.21203/rs.3.rs-7083215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7083215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe cerebrospinal fluid (CSF) surrounding the optic nerve (to differentiate with the intracranial CSF, it is named as optic nerve CSF) plays important roles in nutrient exchange, waste removal, and maintenance of optic nerve function. Recent studies have suggested that the optic disc edema observed in astronauts may be linked to the retention of CSF around the optic nerve, and this retention may even be derived from impaired drainage of optic nerve CSF. However, how the optic nerve CSF is drained out remains inconclusive. We speculated that the optic nerve CSF may be drained out through lymphatic vessels of the optic nerve sheath. Here, we found by immunofluorescence and in vivo fluorescence imaging that, in both rats and humans, the optic nerve sheath has lymphatic vessels. Furthermore, we observed that the pressure in the optic nerve CSF is lower than the intracranial pressure, and the optic nerve CSF does not reflux into the intracranial space but is, instead, drained out into the deep cervical lymph nodes (dcLNs) through the lymphatic vessels in the optic nerve sheath. These observations have significant implications for understanding the physiological turnover and drainage pathways of optic nerve CSF. It may also help better understand the pathogenesis underlying spaceflight and Spaceflight Associated Neuro-Ocular Syndrome (SANS).\u003c/p\u003e","manuscriptTitle":"Optic Nerve Cerebrospinal Fluid Drained-out via Its Sheath Lymphatic Vessels","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-27 10:59:22","doi":"10.21203/rs.3.rs-7083215/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"04cc18d8-5c22-4f0f-b348-9443597744bf","owner":[],"postedDate":"July 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51875454,"name":"Health sciences/Diseases"},{"id":51875455,"name":"Health sciences/Medical research"},{"id":51875456,"name":"Health sciences/Neurology"},{"id":51875457,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-08-24T18:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-27 10:59:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7083215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7083215","identity":"rs-7083215","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}