Ceramide is a bioactive sphingolipid involved in numerous cellular processes. In addition to being the precursor of complex sphingolipids, ceramides can act as second messengers, especially when they are generated at the plasma membrane of cells. Its metabolic dysfunction may lead to or be a consequence of an underlying disease. Recent reports on transcriptomics and electrospray ionization mass spectrometry analysis have demonstrated the variation of specific levels of sphingolipids and enzymes involved in their metabolism in different neurodegenerative diseases. In the present review, we highlight the most relevant discoveries related to ceramide and neurodegeneration, with a special focus on Parkinson’s disease.

Ischemic stroke, caused by the interruption of blood flow to the brain and subsequent neuronal death, represents one of the main causes of disability in developed countries. Therapeutic methods such as recanalization approaches, neuroprotective drugs, or recovery strategies have been widely developed to improve the patient’s outcome; however, important limitations such as a narrow therapeutic window, the ability to reach brain targets, or drug side effects constitute some of the main aspects that limit the clinical applicability of the current treatments. Nanotechnology has emerged as a promising tool to overcome many of these drug limitations and improve the efficacy of treatments for neurological diseases such as stroke. The use of nanoparticles as a contrast agent or as drug carriers to a specific target are some of the most common approaches developed in nanomedicine for stroke. Throughout this review, we have summarized our experience of using nanotechnology tools for the study of stroke and the search for novel therapies.

Ischemic stroke, caused by the interruption of blood flow to the brain and subsequent neuronal death, represents one of the main causes of disability in worldwide. Although reperfusion therapies have shown efficacy in a limited number of patients with acute ischemic stroke, neuroprotective drugs and recovery strategies have been widely assessed, but none of them have been successful in clinical practice. Therefore, the search for new therapeutic approaches is still necessary. Sphingolipids consist of a family of lipidic molecules with both structural and cell signaling functions. Regulation of sphingolipid metabolism is crucial for cell fate and homeostasis in the body. Different works have emphasized the implication of its metabolism in different pathologies, such as diabetes, cancer, neurodegeneration, or atherosclerosis. Other studies have shown its implication in the risk of suffering a stroke and its progression. This review will highlight the implications of sphingolipid metabolism enzymes in acute ischemic stroke.

Recanalization of the occluded artery is the gold standard treatment for acute ischemic stroke, which includes enzymatic fibrinolytic treatment with the use of recombinant tissue plasminogen activators (rtPAs) to disrupt the occluding clot, the use of mechanical thrombectomy to physically remove the clot, or a combination of both. Fibrin is one of the main components of blood clots causing ischemic stroke and is the target of rtPA upon activation of plasminogen in the clot. In addition, fibrin content also influences the efficacy of mechanical thrombectomy. Current imaging methods can successfully identify occlusions in large vessels; however, there is still a need for contrast agents capable of visualizing small thrombi in ischemic stroke patients. In this work, we describe the synthesis and the in vitro characterization of a new diagnostic nanoparticle, as well as the in vivo evaluation in an animal model of thromboembolic stroke. Gd-labeled KCREKA peptides were synthesized and attached onto the surface of PEGylated superparamagnetic nanoparticles. Magnetic resonance imaging (MRI) of blood clots was performed in vitro and in vivo in animal models of thromboembolic stroke. KCREKA-NPs were synthesized by attaching the peptide to the amino (N) termini of the PEG-NPs. The sizes of the nanoparticles, measured via DLS, were similar for both KCREKA-NPs and PEG-NPs (23 ± 4 nm, PDI = 0.11 and 25 ± 8 nm, PDI = 0.24, respectively). In the same line, r₂ relaxivities were also similar for the nanoparticles (149 ± 2 mM Fe s⁻¹ and 151 ± 5 mM Fe s⁻¹), whereas the r₁ relaxivity was higher for KCREKA-NPs (1.68 ± 0.29 mM Fe s⁻¹ vs. 0.69 ± 0.3 mM Fe s⁻¹). In vitro studies showed that blood clots with low coagulation times were disrupted by rtPA, whereas aged clots were almost insensitive to the presence of rtPA. MRI in vitro studies showed a sharp decrease in the T₁ × T₂ signals measured for aged clots incubated with KCREKA-NPs compared with fresh clots (47% [22, 80] to 26% [15, 51]). Furthermore, the control blood showed a higher value of the T₁ × T₂ signal (39% [20, 61]), being the blood clots with low coagulation times the samples with the lowest values measured by MRI. In vivo studies showed a significant T₁ × T₂ signal loss in the clot region of 24% after i.v. injection of KCREKA-NPs. The thrombus age (2.5% ± 6.1% vs. 81.3% ± 19.8%, p < 0.01) confirmed our ability to identify in vivo fresh blood clots. In this study, we developed and tested a dual MRI nanoparticle, acting as T₁ and T₂ contrast agents in MRI analyses. The developed KCREKA-NPs showed affinity for the fibrin content of blood clots, and the MRI signals provided by the nanoparticles showed significant differences depending on the clot age. The developed KCREKA-NPs could be used as a tool to predict the efficacy of a recanalization treatment and improve the triage of ischemic stroke patients.

Sham control groups are essential in experimental animal studies to reduce the influence of surgical intervention. The intraluminal filament procedure is one of the most common models of middle cerebral artery occlusion (MCAO) used in the study of brain ischemia. However, a sham group is usually not included in the experimental design of these studies. In this study, we aimed to evaluate the relevance of the sham group by analyzing and comparing the brain protein profiles of the sham and MCAO groups. In the sham group, 98 dysregulated proteins were detected, compared to 171 in the ischemic group. Moreover, a comparative study of protein profiles revealed the existence of a pool of 57 proteins that appeared to be dysregulated in both sham and ischemic animals. These results indicated that the surgical procedure required for the intraluminal occlusion of the middle cerebral artery (MCA) induces changes in brain protein expression that are not associated with ischemic lesions. This study highlights the importance of including sham control groups in the experimental design, to ensure that surgical intervention does not affect the therapeutic target under study.

Hypertension is a modifiable risk factor for cerebral small vessel disease (cSVD) which leads to blood-brain barrier (BBB) dysfunction. This study used gadolinium-enhanced MRI T₁ and T₂ in stroke-prone renovascular hypertensive rats (RHRSP) to quantify BBB leakage and brain lesions. Serum inflammation and endothelial disruption biomarkers were assessed. Brain aquaporin 4 (AQ4) and retinal morphology were evaluated by histology. RHRSP showed higher systolic blood pressure from the first post-surgical week (134.1 ± 16.6 vs 113.5 ± 11.4 mmHg; p = 0.041). Gadolinium extravasation was increased in the whole brain of RHRSP (p < 0.05), observing cerebral lesions in 50 % of them. Regarding biomarkers, TNF-α was increased (13.5 ± 2.2 vs 11.1 ± 3.5 pg/ml, p = 0.031) while soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) was lower (14.5 ± 2.2 vs 18.9 ± 2.5 pg/ml, p < 0.001) in the acute hypertension phase. AQ4 was increased in the RHRSP brain (1.6 ± 0.7 vs 1.0 ± 0.2 NFU; p = 0.001). Both retinal nuclear layers were reduced in RHRSP (p < 0.05). Gadolinium-enhanced MRI allows the detection of BBB leakage during the establishment of RHRSP. BBB dysfunction is consistent with increased AQ4 and decreased sTWEAK release. Therefore, this animal model is useful for testing potential treatments at cSVD initial stages.