Brainstem blood brain barrier disruption using focused ultrasound: A demonstration of feasibility and enhanced doxorubicin delivery

Magnetic Resonance Image-guided Focused Ultrasound (MRgFUS) has been used to achieve transient blood brain barrier (BBB) opening without tissue injury. Delivery of a targeted ultrasonic wave causes an interaction between administered microbubbles and the capillary bed resulting in enhanced vessel permeability. The use of MRgFUS in the brainstem has not previously been shown but could provide value in the treatment of tumours such as Diffuse Intrinsic Pontine Glioma (DIPG) where the intact BBB has contributed to the limited success of chemotherapy. Our primary objective was to determine whether the use of MRgFUS in this eloquent brain region could be performed without histological injury and functional deficits. Our secondary objective was to select an effective chemotherapeutic against patient derived DIPG cell lines and demonstrate enhanced brainstem delivery when combined with MRgFUS in vivo. Female Sprague Dawley rats were randomised to one of four groups: 1) Microbubble administration but no MRgFUS treatment; 2) MRgFUS only; 3) MRgFUS + microbubbles; and 4) MRgFUS + microbubbles + cisplatin. Physiological assessment was performed by monitoring of heart and respiratory rates. Motor function and co-ordination were evaluated by Rotarod and grip strength testing. Histological analysis for haemorrhage (H & E), neuronal nuclei (NeuN) and apoptosis (cleaved Caspase-3) was also performed. A drug screen of eight chemotherapy agents was conducted in three patient-derived DIPG cell lines (SU-DIPG IV, SU-DIPG XIII and SU-DIPG XVII). Doxorubicin was identified as an effective agent. NOD/SCID/GAMMA (NSG) mice were subsequently administered with 5 mg/kg of intravenous doxorubicin at the time of one of the following: 1) Microbubbles but no MRgFUS; 2) MRgFUS only; 3) MRgFUS + microbubbles and 4) no intervention. Brain specimens were extracted at 2 h and doxorubicin quantification was conducted using liquid chromatography mass spectrometry (LC/MS). BBB opening was confirmed by contrast enhancement on T1-weighted MR imaging and positive Evans blue staining of the brainstem. Normal cardiorespiratory parameters were preserved. Grip strength and Rotarod testing demonstrating no decline in performance across all groups. Histological analysis showed no evidence of haemorrhage, neuronal loss or increased apoptosis. Doxorubicin demonstrated cytotoxicity against all three cell lines and is known to have poor BBB permeability. Quantities measured in the brainstem of NSG mice were highest in the group receiving MRgFUS and microbubbles (431.5 ng/g). This was significantly higher than in mice who received no intervention (7.6 ng/g). Our data demonstrates both the preservation of histological and functional integrity of the brainstem following MRgFUS for BBB opening and the ability to significantly enhance drug delivery to the region, giving promise to the treatment of brainstem-specific conditions

IVIg inhibits MOG_35–55-induced pro-inflammatory cytokine production in IL-11Rα^−/− mice with the exception of IL-17A in the draining lymph nodes.

<p>IL-11Rα^+/+ littermates (WT) and IL-11Rα^−/− mice were immunized with MOG_35–55/CFA (no pertussis toxin), and were injected with either HSA or IVIg (1 g/kg, i.p.) daily. On day 6 (<b><i>D</i></b>) or on day 12 post-immunization (<b><i>A–C</i></b>), spleens and draining lymph nodes were collected, were processed into a single cell suspension, and were cultured with MOG_35–55 (5 µg/ml). <b><i>A</i></b>, The productions of IFN-γ, TNF-α, IL-2 and IL-17A were measured in supernatants of lymph node and splenocyte cultures of IVIg- or HSA-treated IL-11Rα^+/+ (WT) and IL-11Rα^−/− mice. Values are means + SEM of triplicate cultures. These data are representative of three individual experiments. <b><i>B</i></b>, The proliferation in counts per minute (cpm) of spleen cells was measured using a [H³]-thymidine incorporation assay. Shown is the stimulation index, which is the mean + SEM cpm in the MOG_35–55-stimulated wells divided by the cpm in the media control wells. Values are representative of means + SEM of individual mice (N = 4–5/group) in one representative experiment. <b><i>C</i></b>, Sera was taken from mice at 12 days post-immunization and the levels of IL-17 were measured using FlowCytomix assays. Values are means + SEM of individual mice (N = 4–5/group). <b><i>D</i></b>, Lymph node and spleen mononuclear cells taken at day 6 post-immunization (peak of ICOS expression) were stained for CD4, CD25, FoxP3, and ICOS. The frequency of Tregs was determined, as was the frequency of these cells that were ICOS positive. Values are means + SEM of N = 3 individual mice per group. In all cases, groups were compared using a one-way ANOVA and Tukey post-hoc test. *p<0.05 indicates a difference of the IVig group from the HSA-treated, genotype-matched counterpart. #indicates a significant difference of the IL-11Rα^−/− group from the IL-11Rα^+/+, treatment matched counterpart.</p

Clinical Features of EAE in IL-11Rα^+/+ and IL-11Rα^−/− mice treated with IVIg or HSA.

<p>Values for day of onset, cumulative score are means (SEM). Values for peak score are shown as median (25 percentile). Between-group peak score was analyzed using a Kruskal Wallis test. The day of onset and cumulative score features were analyzed using a one-way ANOVA and Tukey post-hoc test. A chi-square test was used to analyze whether disease incidence differed between IVIg and HSA counterparts.</p><p>*indicates a significant difference (p<0.05) from the HSA counterpart. Sample sizes (N) are as indicated.</p

Characterization of the T Cell Compartment in IL-11Rα^+/+ and IL-11Rα^−/− Mice.

<p>Shown are mean (SEM) of values obtained in 3–4 individual age-matched female mice. No measures were found to be significantly different (p<0.05) between IL-11Rα^+/+ and IL-11Rα^−/− mice using a two-tailed Mann-Whitney U or T-test.</p

IVIg inhibits EAE by preventing the expansion and Th1 and Th17 cytokine production by MOG_35–55 reactive T cells and by increasing ICOS expression by Tregs.

<p>EAE was induced in female C57BL/6J mice by immunization with MOG_35–55 and CFA plus pertussis toxin and mice were administered daily i.p. injections of IVIg or 1×PBS beginning at day of disease induction (N = 12/group). At 10 post-immunization, spleens were collected from N = 2/mice per group and were pooled for ex vivo stimulation with MOG_35–55. Remaining mice (N = 10/group) were followed for clinical signs. <b><i>A</i></b>, shows the mean + SEM severity of clinical signs of mice in each group over the period of observation. <b><i>B</i></b>, shows the levels of IL-2 (at 48 h), IFN-γ, TNF-α and IL-17A (at 72 h) in splenocyte cultures as measured by ELISA. Values are means + SEM of triplicate cultures in one representative experiment. <b><i>C</i></b>, shows proliferation of cells in triplicate splenocyte cultures in response to MOG_35–55 (5 µg/ml) was measured in counts per minute (cpm) and was expressed relative to the cpm in the non-peptide-containing wells. This ratio is the stimulation index. In <b><i>A–C</i></b>, data are representative of 2–3 independent experiments. <b><i>D–G</i></b>, represents a separate study where C57BL6/J mice were immunized with MOG_35–55/CFA (without pertussis toxin) and were treated with PBS or IVIg daily. After 4, 6, 8, or 10 days, spleens were harvested, were processed into a single cell suspension for the determination of CD4, CD25, FoxP3, and ICOS using flow cytometry. <b><i>D</i></b> shows the frequency and number of CD4⁺CD25⁺FoxP3⁺ cells at these different time points after immunization. Values are means ± SEM 3 individual mice per group at each time point. <b><i>E</i></b> shows representative staining of FoxP3 and CD25 in the live, CD4⁺ gate while <b><i>G</i></b> shows representative ICOS staining of the live CD4⁺CD25⁺FoxP3⁺ population at day 6 post-immunization (peak of ICOS expression, data not shown). <b><i>F</i></b> Shows the mean ± SEM percent of Tregs that were positive for ICOS staining. Values are means + SEM percentages obtained in 3 individual mice per group. ***p<0.001, **p<0.01, *p<0.05 as determined using a t-test or Mann-Whitney U test.</p

IVIg treatment induces a profound increase in the circulating levels of IL-11 in mice.

<p><b><i>A, B,</i></b> Individual C57BL6/J mice were administered IVIg (2.0 g/kg) or PBS. <b><i>A</i></b>, Serum was collected for IL-11 protein measurement using ELISA at 0, 3, 6, 12, 18 and 24 hours post-injection. *indicates a significant difference (*p<0.05) from time zero as determined using a one-way ANOVA and a Tukey post-hoc test. <b><i>B</i></b>, Tissues (spleen, liver, bone marrow, and pooled axillary and inguinal lymph nodes) were collected from mice at 6 h post-injection, total RNA was isolated and was reverse-transcribed to cDNA, and IL-11 mRNAs were measured in samples using real-time qRT-PCR. Shown is the abundance of IL-11 mRNAs (normalized to beta-actin) and expressed as fold-change relative to the comparator sample (bone marrow sample from PBS-treated mouse). *indicates a difference from PBS-injected group as determined using a Student’s t-test assuming equal variances (two-tailed, *p<0.05). <b><i>A & B</i></b> show means + SEM of values obtained from individual mice. <b><i>C</i></b>, EAE was induced in C57BL/6J mice with MOG_35–55/CFA and pertussis toxin. Mice were injected with IVIg or HSA daily (1 g/kg, i.p.). Shown are the IL-11 levels (pg/ml) in the sera of mice at 6 h post-injection on the day of onset (0), and at 10, 17, and day 45 post-immunization with MOG_35–55 and CFA. Values are means ± SEM of readings from 2–5 individual mice/group/time point. *indicates a significant difference (p<0.05) from the HSA-injected, genotype-matched counterpart. <b><i>D</i></b>, shows a heat map of serum cytokine/chemokine levels taken at 6 h post IVIg or PBS injection with N = 8 mice/group. Each data box in the heat map represents a reading from an individual mouse.</p

IL-11 inhibits the proliferation and IL-17A production by lymph node cells under Th17-polarizing conditions.

<p>Lymph node cells were harvested from C57BL/6J mice (N = 3/group). They were dissociated into a single-cell suspension and were stimulated with plate-bound anti-CD3 and anti-CD28 (0.5 µg/ml) in the absence of added cytokines (Th0) or in the presence of IL-6 (30 ng/ml), TGF-β (3 ng/ml) and anti-IFN-γ neutralizing antibody (10 µg/ml). The proliferation of (<b><i>A</i></b>) and IL-17A (<b><i>B</i></b>) and IFN-γ (<b><i>C</i></b>) cytokine production by lymph node cells was measured using H³-thymidine incorporation and ELISA assays, respectively. Values represent mean + SEM pg/ml or cpm in triplicate cultures. Results are representative of two individual experiments. *indicates a significant (p<0.05) difference from the 0 ng/ml concentration group as determined using a one-way ANOVA and Tukey post-hoc test. Note that IFN-γ was not detected under Th17 skewing conditions and is not shown.</p

IL-11Rα^−/− mice are more resistant to the protective effects of IVIg treatment during EAE.

<p>EAE was induced in C57BL/6J IL-11Rα^+/+ littermates (WT) or IL-11Rα^−/− mice that were administered IVIg or HSA (1 g/kg) daily beginning on the day of EAE induction. Mice were followed for clinical signs and a histological analysis of inflammation and demyelination was conducted at the end-point of the experiment. <b><i>A</i></b>, shows the mean + SEM clinical scores of mice over a time-course of EAE. The graph shows combined data from three consecutive EAE studies that each contained (N = 5–10 mice/group). <b><i>B</i></b>, shows representative spinal cord sections from control- or IVIg-treated IL-11Rα^−/− or WT mice stained with haematoxylin and eosin and luxol fast blue. <b><i>C</i></b>, shows the percent quadrants of spinal cords (N = 10 sections/mouse) that were positive for meningitis or perivascular cuffs in EAE mice. <b><i>D</i></b>, shows the percent demyelination in these spinal cord sections. Values in <b><i>C & D</i></b> are means + SEM of N = 4–6 mice per group for one representative experiment. *represents a significant difference (*p<0.05) from the HSA-treated, genotype-matched counterpart as determined by one-way ANOVA and a Tukey post-hoc test.</p