Establishment Of A Mouse Model For Leptomeningeal Carcinomatosis

Objectives: Leptomeningeal carcinomatosis(LC)is a rare type of central nervous system metastases of various solid tumors,characterized by a focal or diffuse infiltration of malignant tumor cells to the leptomeninges and subarachnoid space,and spreading throughout the cerebrospinal fluid(CSF).Most of the primary tumors derive from lung cancer and adenocarcinoma is the most common histological type.The incidence of LC is increasing over the past few years,but the treatment effects are still unsatisfying,which is partly due to the lack of precise treatment of different types or various pathological feature of primary tumors.In order to investigate more effective therapeutic targets for LC and evaluate the efficacy and safety of different drugs or administration methods,an experimental animal model of LC should be established first.There have been few reports on animal experiments of LC so far.Researchers abroad conducted studies in early years on LC rats model via puncture of cisterna magna(CM).Intrathecal injection requiring complicated process.In the present study,we take a modified method of CM injection,aiming to make an easy and reproducible mouse model for LC.In addition,we investigate the expression of CD31 on leptomeninges in this mouse model.Methods:1 Injection into the cisterna magna in miceTwenty female C57BL/6 mice were divided into two groups randomly and injected PBS or cell suspension of Lewis Lung carcinoma cell(1.5×105cells)7.5?l into CM respectively.The dynamic change of body weights in mice after CM injection were recorded and the neurological manifestations were observed and evaluated.In addition,another 15 mice were divided into groups of control(n=3),PBS(n=6)and LLC(n=6).Mice that had obvious symptoms were sacrificed for perfusion with 4%paraformaldehyde,then the tissue sections of brain and spinal cord were used for hematoxylin-eosin staining or immunohistochemical analysis.2 HE staining for histopathological evaluationTissue specimens of brain and spinal cord fixed by 4% paraformaldehyde were routinely paraffin-embedded.For histopathological analysis on pia mater of brain and spinal cord,5-?m sections were made using a paraffin slicing machine(Leica RM2235)to stain with HE.3 ImmunohistochemistryImmunohistochemical analysis of CD31 expression in the area of vessels in meninges of pons in mice of PBS group and LLC model group were carried out for assessment the tumor angiogenesis of LC.Results:1 The distribution of ink in the mice subarachnoid space soon after CM injection was observed.The dye was found mostly in the CM cistern and the ventral cisterns.2 Behavioral observation of mice in two groups after CM injection of LLC or PBS(1)The dynamic change of mice body weights in two groups inoculated with 7.5?l LLC cell suspension or PBS respectively were recorded.On the first day after inoculation,mice in two groups exhibited significant weight loss,PBS-injected control mice were(17.02±1.63)g,LLC-injected mice were(17.08±1.06)g,there was no statistical significance between the two groups(P=0.921).On the day 2 and day 3,the body weights of mice were still low.From day 4 to day 10,the mice body weights gained slowly in both groups,showing no significant differences between groups(P?0.05).On the day 11,mice in LLC group showed progressive weight loss till death,while in the PBS group,the body weights still increased slowly(P=0.029).Mice in LLC model group all(n=10)died within 40 days,the median survival time was32.5d,while in the PBS group,all mice(n=10)survived till endpoint(P?0.0001).(2)LLC-injected mice developed symptoms on the day 10 ~ day16,with an average of(13±2)d.All mice(n=10)in this group showed lethargy or poor general condition(10/10),6 of whom showed an arched back posture with a stretched neck(6/10),5 of whom showed unsteady gait(5/10)and the manifestation of rotatory movements when lifted by the tail appeared in 2mice(2/10).Mice in PBS group showed normal activities,no such symptoms were observed.3 HE staining for histopathology showed a diffuse infiltration of malignant tumor cells to the leptomeninges and subarachnoid space in mice with LLC injection,tumor cells were mainly observed in the base of the brain and the cervical cord.4 Immunohistochemical analysis of CD31 expression in the area of vessels in mice meninges showed a significant difference between the arachnoidal vascular density in the control mice(6.08±1.02 vessels mm-1 of arachnoid lining)and in the mice with LLC injection(12.83 ±3.01 vessels mm-1;P=0.021).Conclusions:1 It is feasible to develop a mouse model for LC with Lewis lung carcinoma cell injection into the cisterna magna.2 Mice with LC may show symptoms during progression of the disease,such as lethargy or poor general condition,an arched back posture with a stretched neck,unsteady gait,and the manifestation of rotatory movements when lifted by the tail,but not all mice have the same symptom.3 High CD31 expression in the area of vessels in meninges indicates the increased arachnoidal vascular density in mice with LC and that neovascularization may have a important role in the growth of LC in mice.