cervical-lymph-duct-cannulated-rat-model-for-assessing-lymphatic

Cervical Lymph Duct-Cannulated Rat Model for Assessing Lymphatic Transport from the Head and Brain

Lymph flows from the central nervous system (CNS) via a series of lymphatic vessels (LVs) and lymph nodes that converge at the cervical lymph ducts. In ...

Monash University

To examine the adverse drug reactions (ADRs) and the efficacy of intravenous iron polymaltose and ferric carboxymaltose (FCM) among gynecology/obstetric patients with anemia.

Retrospective cohort study comparing the adverse reactions and efficacy of intravenous iron polymaltose with ferric carboxymaltose for iron deficiency anemia.

Dietary lipids, highly lipophilic drugs, antigens and immune cells are transported from the intestine to the mesenteric lymph nodes (MLNs) via mesenteric lymphatic vessels. Recently our lab reported that the mesenteric lymphatic vessels become highly branched and leak lymph to the surrounding mesenteric adipose tissue (MAT) in mice and humans with obesity, promoting insulin resistance. This study aimed to investigate the impact of obesity-associated mesenteric lymph leakage on the trafficking of a dietary lipid (oleic acid), lipophilic drug (cyclosporin A) and antigen (ovalbumin) from the intestine to MLNs. C57BL/6J mice were fed a control fat diet (CFD), or a high fat diet (HFD) for up to 35 weeks leading to obesity and impaired glucose tolerance. C-oleic acid, H-cyclosporin or Cy5.5-ovalbumin were administered orally, and blood plasma and tissues collected to measure radioactivity or fluorescence levels. The accumulation of C-oleic acid, H-cyclosporin and Cy5.5-ovalbumin in MAT was significantly increased in HFD compared to CFD fed mice, whereas in the MLNs there was less accumulation (H-cyclosporin and Cy5.5-ovalbumin) or no significant difference (for C-oleic acid). The mass ratio of these molecules in MLNs compared to MAT was thus significantly decreased. Obesity-associated mesentery lymph leakage appears to divert dietary lipids, lipophilic drugs and antigens away from their normal lymphatic trafficking pathways from the intestine to MLNs and instead results in leakage into MAT. This is likely to contribute to known detrimental changes to lipid metabolism, immunotherapy and mucosal immunity in obesity.

Obesity-associated mesenteric lymph leakage impairs the trafficking of lipids, lipophilic drugs and antigens from the intestine to mesenteric lymph nodes.

 Fluids, solutes and immune cells have been demonstrated to drain from the brain and surrounding structures to the cervical lymph vessels and nodes in the neck  meningeal lymphatics, nasal lymphatics and/or lymphatic vessels associated with cranial nerves. A method to cannulate the efferent cervical lymph duct for continuous cervical lymph fluid collection in rodents has not been described previously and would assist in evaluating the transport of molecules and immune cells from the head and brain  the lymphatics, as well as changes in lymphatic transport and lymph composition with different physiological challenges or diseases.  To develop a novel method to cannulate and continuously collect lymph fluid from the cervical lymph duct in rats and to analyze the protein, lipid and immune cell composition of the collected cervical lymph fluid.  Male Sprague-Dawley rats were cannulated at the carotid artery with or without cannulation or ligation at the cervical lymph duct. Samples of blood, whole lymph and isolated lipoprotein fractions of lymph were collected and analyzed for lipid and protein composition using commercial kits. Whole lymph samples were centrifuged and isolated pellets were stained and processed for flow cytometry analysis of CD3, CD4, CD8a, CD45R (B220) and viable cell populations.  Flow rate, phospholipid, triglyceride, cholesterol ester, free cholesterol and protein concentrations in cervical lymph were 0.094 ± 0.014 mL/h, 0.34 ± 0.10, 0.30 ± 0.04, 0.07 ± 0.02, 0.02 ± 0.01 and 16.78 ± 2.06 mg/mL, respectively. Protein was mostly contained within the non-lipoprotein fraction but all lipoprotein types were also present. Flow cytometry analysis of cervical lymph showed that 67.1 ± 7.4% of cells were CD3/CD4 T lymphocytes, 5.8 ± 1.6% of cells were CD3/CD8 T lymphocytes, and 10.8 ± 4.6% of cells were CD3/CD45R B lymphocytes. The remaining 16.3 ± 4.6% cells were CD3/CD45 and identified as non-lymphocytes.  Our novel cervical lymph cannulation method enables quantitative analysis of the lymphatic transport of immune cells and molecules in the cervical lymph of rats for the first time. This valuable tool will enable more detailed quantitative analysis of changes to cervical lymph composition and transport in health and disease, and could be a valuable resource for discovery of biomarkers or therapeutic targets in future studies.

Development and application of a novel cervical lymph collection method to assess lymphatic transport in rats.

In recent years, the drainage of fluids, immune cells, antigens, fluorescent tracers, and other solutes from the brain has been demonstrated to occur along lymphatic outflow pathways to the deep cervical lymph nodes in the neck. To the best of our knowledge, no studies have evaluated the lymphatic transport of therapeutics from the brain. The objective of this study was to determine the lymphatic transport of model therapeutics of different molecular weights and lipophilicity from the brain using cervical lymph cannulation and ligation models in rats. To do this, anesthetized Sprague-Dawley rats were cannulated at the carotid artery and cannulated, ligated, or left intact at the cervical lymph duct. Rats were administered C-ibuprofen (206.29 g/mol, logP 3.84), H-halofantrine HCl (536.89 g/mol, logP 8.06), or H-albumin (∼65,000 g/mol) via direct injection into the brain striatum at a rate of 0.5 μL/min over 16 min. Plasma or cervical lymph samples were collected for up to 6-8 h following dosing, and brain and lymph nodes were collected at 6 or 8 h. Samples were subsequently analyzed for radioactivity levels via scintillation counting. For C-ibuprofen, plasma concentrations over time (plasma AUC) were >2 fold higher in lymph-ligated rats than in lymph-intact rats, suggesting that ibuprofen is cleared from the brain primarily via nonlymphatic routes (e.g., across the blood-brain barrier) but that this clearance is influenced by changes in lymphatic flow. For H-halofantrine, >73% of the dose was retained at the brain dosing site in lymph-intact and lymph-ligated groups, and plasma AUC values were low in both groups (<0.3% dose.h/mL), consistent with the high retention in the brain. It was therefore not possible to determine whether halofantrine undergoes lymphatic transport from the brain within the duration of the study. For H-albumin, plasma AUC values were not significantly different between lymph-intact, lymph-ligated, and lymph-cannulated rats. However, >4% of the dose was recovered in cervical lymph over 8 h. Lymph/plasma concentration ratios of H-albumin were also very high (up to 53:1). Together, these results indicate that H-albumin is transported from the brain not only via lymphatic routes but also via the blood. Similar to other tissues, the lymphatics may thus play a significant role in the transport of macromolecules, including therapeutic proteins, from the brain but are unlikely to be a major transport pathway from the brain for small molecule drugs that are not lipophilic. Our rat cervical lymph cannulation model can be used to quantify the lymphatic drainage of different molecules and factors from the brain.

Quantifying the Lymphatic Transport of Model Therapeutics from the Brain in Rats.

The drainage of fluid and solutes along lymphatic pathways from the brain has been found to be impaired in mouse models of multiple sclerosis, Alzheimer's disease, and Parkinson's disease where neuroinflammation is present. We recently demonstrated that H-albumin, a model therapeutic protein (∼65 kDa), undergoes preferential lymphatic transport from the brain using a cervical lymph cannulation model in healthy rats. We thus hypothesized that neuroinflammation would impede the lymphatic transport of H-albumin from the brain. Our aim was to quantify the impact of acute neuroinflammation on drainage of the model therapeutic protein (H-albumin) from the rat brain into blood and deep cervical lymph. To establish the required neuroinflammation model, male Sprague-Dawley rats were administered an intraperitoneal (IP) dose of 0.5-2 mg/kg lipopolysaccharide (LPS, ) or a saline control. After 12 or 24 h, brain samples were collected and analyzed for concentrations of interferon gamma (IFN-γ) using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The impact of neuroinflammation on the drainage of H-albumin from the brain was determined via IP administration of 2 mg/kg LPS or saline followed by cannulation of the carotid artery for blood collection 24 h later with/without cannulation or ligation at the efferent deep cervical lymph trunk. Rats were then administered H-albumin via direct injection into the brain striatum or via intravenous (IV) injection (lymph-intact group only). Blood ± lymph samples were collected for up to 8 h following dosing. At the end of the study, brain and lymph node samples were harvested for biodistribution analysis, with samples analyzed for radioactivity levels via scintillation counting. Brain concentrations of the pro-inflammatory cytokine IFN-γ were only significantly elevated 24 h after IP administration of 2 mg/kg LPS compared to saline control. Therefore, this induction regimen was utilized for subsequent studies. The plasma concentrations of H-albumin over time were elevated in LPS-induced rats compared to saline-injected rats in the lymph-intact and lymph-ligated groups but not in the lymph-cannulated group. In the deep cervical lymph-cannulated animals, the lymph transport of H-albumin was not increased and appeared to be slower in the LPS-administered rats. Acute LPS-induced neuroinflammation therefore led to an enhanced overall transport of H-albumin from the brain into the systemic circulation. This appeared to be primarily due to increased transport of H-albumin from the brain directly into the blood circulation as H-albumin transport from the brain via the lymphatics was not increased in the LPS-induced neuroinflammation model. Such changes in the clearance of therapeutic proteins from the brain in the setting of neuroinflammation may impact the therapeutic efficacy and safety.

Article	Year
Retrospective cohort study comparing the adverse reactions and efficacy of intravenous iron polymaltose with ferric carboxymaltose for iron deficiency anemia. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics\| PubMed ID: 29498039	2018
Obesity-associated mesenteric lymph leakage impairs the trafficking of lipids, lipophilic drugs and antigens from the intestine to mesenteric lymph nodes. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V\| PubMed ID: 36283633	2022
Development and application of a novel cervical lymph collection method to assess lymphatic transport in rats. Frontiers in pharmacology\| PubMed ID: 36744256	2023
Quantifying the Lymphatic Transport of Model Therapeutics from the Brain in Rats. Molecular pharmaceutics\| PubMed ID: 38579335	2024
Acute Neuroinflammation Alters the Transport of a Model Therapeutic Protein from the Brain into Lymph and Blood. Molecular pharmaceutics\| PubMed ID: 39185947	2024

Thu Hoang

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