A Retro-orbital Sinus Injection Mouse Model to Study Early Events and Reorganization of the Astrocytic Network during Pneumococcal Meningitis.

Pneumococcal (PN) meningitis is a life-threatening disease with high mortality rates that leads to permanent neurological sequelae. Studies of the process of bacterial crossing of the blood brain barrier (BBB) are hampered by the lack of relevant in vitro and in vivo models of meningitis that recapitulate the human disease. PN meningitis involves bacterial access to the bloodstream preceding translocation across the BBB. A large number of PN meningitis models have been developed in mice, with intravenous administration via the lateral tail vein representing the main way to study BBB crossing by PN. While in humans, meningitis is not always associated with bacteremia, PN meningitis after intravenous injection in mice usually develops following sustained and very high bacteremic titers. High grade bacteremia, however, is known to favor inflammation and BBB permeabilization, thereby increasing PN translocation across the BBB and associated damages. Therefore, specific processes associated with early events of PN translocation may be blurred by overall changes in the inflammatory environment and potentially systemic dysfunction in the case of severe sepsis. Here, we report a mouse meningitis model induced by PN injection in the retro-orbital (RO) sinus. We show that, in this model, mice appear to control bacteremic levels during the first 13 h post-infection, while PN crossing of the BBB can be clearly detected by fluorescence confocal microscopy analysis of brain slices as early as 6 h post-infection. Because of the low frequency of events, however, PN translocation across brain parenchymal vessels at early time points requires a rigorous and systematic examination of the brain volume.

Peripheral Nerve Microscopic Changes Related to Study Procedures: Two Nonclinical Case Studies.

Peripheral nerves are routinely examined microscopically during the nonclinical safety assessment of therapeutics. In addition to test article-related on- or off-target changes, microscopic changes in peripheral nerves may also be caused by study procedures, such as parenteral test article administration and blood or tissue sampling. We present 2 nonclinical case studies in which nonstandard peripheral nerves had study procedure-related histologic changes. The first case study describes mouse trigeminal nerve changes as a result of blood sampling via retro-orbital sinus puncture. These changes included minimal-to-mild nerve fiber (axonal) degeneration associated with macrophage infiltration. The second case study presents rat brachial plexus changes associated with animal handling and blood sampling. Brachial plexus changes included minimal-to-moderate inflammation, focal hemorrhage, and nerve fiber degeneration. In both cases, the histological changes were morphologically indistinguishable from those that might be due to test article. Therefore, careful consideration of the incidence and severity across groups and a review of study procedures to rule out handling-related nerve damage are essential before identifying a test article-related effect on peripheral nerves. Study design considerations to avoid such procedure-related changes will be discussed, as well as sampling strategies to help distinguish these from test article-related effects.

Histological and Electrophysiological Changes in the Retinal Pigment Epithelium after Injection of Sodium Iodate in the Orbital Venus Plexus of Pigmented Rats.

PURPOSE: To characterize histopathologic and electroretinographic (ERG) changes in the retina of pigmented rats injected with sodium iodate in order to establish a model of retinal degeneration for future cell therapy studies. METHODS: In 50 male pigmented rats weighing 250-300 grams, NaIO3 was injected into the left orbital venous plexus at 40 and 60 mg/kg doses (25 eyes in each group). Fourteen rats received phosphate buffered saline (PBS) injection in their left orbital plexus and were considered as the sham-control group. Histopathologic and ERG studies were performed at baseline and on days 1, 7, 14 and 28 after the injections. RESULTS: Progressive retinal pigment epithelial (RPE) changes were observed from the first day of injection in both the 40 and 60 mg/kg study groups in a dose dependent manner. These changes manifested as loss of melanin pigment and accumulation of lipofuscin in RPE cells with subsequent cell death and patchy loss of RPE cells (in flat mounts), as well as thinning of the outer nuclear layer and later the inner nuclear layer in the succeeding days. ERG showed a progressive and significant decrease in a- and b- wave amplitudes in both case groups relative to baseline values and the controls (P < 0.05). CONCLUSION: NaIO3 injection into the retrobulbar venous plexus of pigmented rats can result in significant and progressive damage to the RPE and subsequently to the neuroretina of the injected eye, and may serve as a model of retinal degeneration.

Humanizing NOD/SCID/IL-2Rγnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34(+) cells.

BACKGROUND: Humanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function. METHODS: Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34(+) hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS: Most of the NSG mice tolerated the regimen well. The percentage of hCD45(+) and CD19(+) cells rose significantly in a time-dependent manner. The median percentage of hCD45(+)cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percentage of hCD45(+) cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19(+) cells in BM at weeks 8 and 12 was 21.5% and 39%, respectively (P=0.04). Similarly, the median percentage of hCD19(+) cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19(+) B cells in PB was 23% at week 12. At week 8, hCD3(+) T cells were barely detectable, while hCD7(+) was detected in the BM and spleen. The percentage of hCD3(+) T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice. CONCLUSION: We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45(+) cells than earlier studies without any significant toxicity. And human CD45(+) cell engraftment at week 8 was comparable to that of week 12.

Humanizing NOD/SCID/IL-2Rγnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34+ cells

BACKGROUND: Humanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function. METHODS: Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS: Most of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+ cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percentage of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, respectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+ B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice. CONCLUSION: We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any significant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.

Comparison of haematopoietic stem cell engraftment through the retro-orbital venous sinus and the lateral vein: alternative routes for bone marrow transplantation in mice.

Bone marrow transplantation in mice is performed by intravenous administration of haematopoietic repopulating cells, usually via the lateral tail vein. This technique can be technically challenging to carry out and may cause distress to the mice. The retro-orbital sinus is a large area where there is a confluence of several vessels that provides an alternative route for intravenous access. Retro-orbital injection, although aesthetically unpleasant, can be performed rapidly without requiring mechanical restriction or heat-induced vasodilation. In addition, this technique can be easily learned by novice manipulators. This route of administration has been reported for use in bone marrow transplantation but there is no comparison of retro-orbital and tail vein injections reported for this specific purpose, although both routes have been compared for many other applications. Here, we provide for the first time a comprehensive comparison between tail vein and retro-orbital injections for two different bone marrow transplant scenarios in P3B and B6D2F1 mice. In both cases, no significant differences regarding donor engraftment were observed between mice transplanted using each of the techniques. Haematological counts and leukocyte subpopulation distribution were practically identical between both animal groups. Moreover, donor engraftment levels were less homogenous when cells were transplanted by tail vein injection, probably due to a higher risk of failure associated with this technique. All these data suggest that retro-orbital injection is a compelling alternative to conventional tail vein injection for bone marrow transplant in mice, providing similar and more homogenous haematopoietic reconstitution.