Summary

Stroke poses a major clinical threat, attributing to 7 % of deaths worldwide. It is the second cause of death globally affecting 17 million people every year, with only one-third of patients surviving with no complications. Stroke can be categorised into major subtypes of different pathophysiology, ischaemic and haemorrhagic stroke. Ischaemic stroke is caused by cerebrovascular vessel occlusion resulting from thrombus or emboli. Haemorrhagic stroke is caused by a rupture of a damaged or abnormal blood vessel leading to local bleeding within the cranium and accounts for 15-20 % of all stroke cases. Although haemorrhagic stroke has a lower incidence rate, outcomes are worse than ischaemic stroke with almost 40 % mortality risk. Haemorrhagic stroke can be further sub-categorised into intracerebral haemorrhage (ICH), subarachnoid and interventricular bleeds depending on the location of the ruptured vessel. The most common of those is ICH, which results from a direct leak of blood into the brain parenchyma due to vascular rupture secondary to hypertension, cerebral amyloid angiopathy, vascular aneurysm, tumour, or predisposing genetic conditions. Age is another un-adjustable risk factor for ICH which doubles the chance of the disease every 10 years of life. The increased use of oral anticoagulant to prevent ischaemic stroke causes another threat and carries a 50 % mortality rate in the occurrence of ICH due to the increased chances of re-bleeding.
Secondary injury is triggered in ICH due to the breakdown of blood products and this has been shown to stimulate several cellular mechanisms which in turn exacerbate the original injury. Extracellular contact with blood components exerts neurotoxic effects from the release of free radicals causing vasogenic oedema. Brain-resident (microglia) and infiltrating immune cells (macrophages) attempt to clean up the haematoma debris, which is the initial driving force of the inflammatory response to stop further inflammation and in parallel, a host of chemokines and cytokines are released. Red blood cell (RBC) lysis also occurs, triggering oxidative stress and free radical formation. Iron toxicity has also been noted to cause microglial activation and proliferation, enhancing the inflammatory response. Any recovery in ICH is thought to be dependent on haematoma resolution, oedema reduction, neuroplastic abilities of the brain and neurogenesis.
Treatments that can salvage damaged brain tissue after ICH are currently lacking. The lack of treatments in ICH is partly due to the complexity of the underlying pathophysiology but also the difficulty in getting enough drug into the injury site due to the protective nature of the blood-brain barrier (BBB). This highlights a real need to develop new strategies to enhance drug delivery to the brain in ICH to increase survival rates and improve the post-stroke quality of life for ICH patients. Under normal conditions, the BBB creates a sterile environment for neurons via tight control of the movement of substances between peripheral blood and neuronal tissues. This interface comprises endothelial cells and the tight junction proteins that regulate paracellular transport, pericytes, astrocytes, and transcellular vesicles which regulate the trafficking of small molecules through the endothelial cells. Maintaining BBB function is vital to maintain normal brain function with damage contributing to a host of neurological pathologies. Paradoxically, the protective nature of BBB also limits the delivery of therapeutic drugs into the brain. However, preclinical data and also human studies have shown that BBB integrity is altered following ICH, which may provide a window of opportunity to deliver drugs effectively into the haematoma and surrounding brain tissue. Therefore, understanding the mechanisms and the timeframe of BBB alteration in ICH is crucial for the development of potential new treatments.
Previously, the authors showed that they can selectively target the lesioned area in ischaemic stroke using lipid nanoparticles (liposomes) by taking advantage of enhanced transcellular and paracellular pathways that contribute to BBB disruption after cerebral ischaemia. Liposomes offer several advantages compared to conventional medications including; a) small size and long circulation half-life that enable selective and efficient lesion targeting, and b) adaptation to encapsulate a wide range of therapeutic molecules and contrast agents which offer an ideal opportunity to image where the drug is being delivered.
In this study, the authors hypothesised that similar selective targeting is possible using liposomes in ICH, with BBB disruption being the primary mechanism by which entrance is permitted. Such an approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for ICH therapy and is not achievable using free small drug molecules due to short circulation half-life. To understand the timing of liposome penetration into the brain in ICH, the authors measured brain liposome accumulation using radioisotope and optical detection methods. To confirm that leakage of liposomes was primarily through a disrupted BBB, the authors used dynamic-contrast enhanced MRI to detect the leakage of MRI contrast agent Gd-DOTA. Similar patterns of leakage were observed strongly indicating that liposome penetrance at acute timepoints (0-72 h post-ICH) is mediated by self-diffusion across the disrupted BBB.

Results from the nanoScan SPECT/CT

For the SPECT-CT imaging, mice were subjected to anaesthesia via the inhalation of 2.5 % isoflurane in a mixture of 30 % oxygen and 70 % nitrous oxide. Each animal was then intravenously injected with 200 ul of the radioactive ¹¹¹In-Lp (8-9 MBq). At different time points after injection (t= 0-1 h, & 24 h) SPECT/CT imaging was carried out using the nanoScan^® SPECT/CT scanner. SPECT images were obtained in 20 projections over 40-60 min using a 4-head scanner with 1.4 mm pinhole collimators. CT scans were taken at the end of each SPECT acquisition using a semi-circular method with a full scan, 480 projections, maximum FOV, 35 kV energy, 300 ms exposure time and 1-4 binning. Acquisitions were done using the Nucline v2.01 (Build 020.0000) software, while the reconstruction of all images and fusion of SPECT with CT images was performed using the Interview™ FUSION bulletin software. The images were further analysed using VivoQuant 3.0 software where the SPECT images with scale bars in MBq were corrected for decay and the slight differences in radioactivity in the injected doses between animals. For a quantitative assessment of ¹¹¹In-Lp in the brain, a cut and count method was used. Mice were anaesthetized by isofluorane inhalation and each mouse was injected via the tail vein with 200 μl containing ¹¹¹In-Lp labelled with approximately 8-9 MBq. 24 h after injection, mice were perfused with iced cold saline (0.9 %) followed by PFA (4 %) to remove any ¹¹¹In-Lp from the blood before brain tissues were collected. Each sample was weighed and counted on a gamma counter (Perkin Elmer, USA), together with a serial dilution of the injected dose. The results were represented as the percentage of the injected dose (% ID / gm tissue ± SEM), n = 4-5 mice per group.
Figure 1. shows the imaging and quantification of liposomes accumulation into the brain after inducing ICH into the right hemisphere A) Schematic presentation of experimental design and the time frame of ¹¹¹In-Lp I.V injection after ICH. Each time point represents a separate group that received a single injection of ¹¹¹In-Lp (8-9 MBq) I.V. Representative SPECT/CT imaging for the same mice over B) the first hour after I.V administration and C) 24 h later confirmed the selective accumulation of the liposomes into the ipsilateral side of the brain (right) compared to the contralateral side. Images shown here are for the same mouse from each group. The scale bar is the same in B and C. Healthy mice (where no ICH was induced) showed no detectable ¹¹¹In-Lp level in the brain. The signal observed in healthy mice is due to the presence of ¹¹¹In-Lp in the blood that is mainly found in the circulation outside the brain. This reduced substantially 24 h after injection of ¹¹¹In-Lp as they start to clear from the blood. D) Quantification of the ¹¹¹In-Lp level in the brain 24 h after I.V injection revealed a biphasic entry pattern with maximum accumulation observed between 3-24 h and 48-72 h post-ICH. Values are expressed as % of I.D ± SEM per gram brain tissue. E) Quantification of ¹¹¹In-Lp levels in the CSF indicated no significant differences in CSF liposomal level compared to healthy mice. The data in D & E were analysed by one-way ANOVA followed by Tukey multiple comparison tests (n = 4-5). The data were considered significant if p values < 0.05.

• This study provides evidence that liposomes can selectively accumulate within the lesion site in a collagenase model of ICH. Liposomal entry into the brain peaked when injected 3 h and 48 h after ICH and correlated with the changes in leakage of Gd-DOTA into the lesion detected using DCE-MRI, indicating the primary mechanism of penetrance was via self-diffusion across the disrupted BBB.
• Liposomal accumulation observed is very specific to the haemorrhage area and showed 40 % co-localisation with activated microglia at the lesion site.
• These observations offer an extremely promising technology for ICH treatment enabling time-specific new therapies and spur the rejuvenation of drugs that have been tested before in ICH but have been missed due to failed efficacy or unwanted peripheral toxicity.

Full article on thno.org