A conversation with Prof. Johannes Boltze, MD, PhD, from the University of Lübeck, Germany, along with co-authors Michel M. Modo, PhD; Jukka Jolkkonen, PhD; and Marietta Zille, PhD, regarding the future of animal modeling for poststroke tissue repair.
Interviewed by Shashank Shekhar, MD, MS, Vascular Neurology Fellow, University of Mississippi Medical Center.
They will be discussing the paper “Future of Animal Modeling for Poststroke Tissue Repair,” published in the May issue of Stroke. The article is part of a Focused Update in Cerebrovascular Disease centered on stem cells and cell-based therapies.
Dr. Shekhar: First of all, I would like to thank Prof. Boltze and his co-authors for agreeing to do this interview. This is a very interesting paper where you have not only summarized the current animal research in tissue restoration and future trajectories in animal research for post-stroke repair, but also provided important strategies to overcome the hurdles in implementing successful and clinically relevant animal models.
Could you tell the readers why studying pre-clinical animal models for post-stroke tissue repair is important?
Dr. Boltze: True tissue repair, if it was achieved, will be a highly complicated endeavor that presumably requires numerous individual steps and the targeted modification of processes in the lesioned brain. Some of these processes may be currently unknown. Sophisticated in vitro systems, such as brain organoids, may be used to design intervention strategies towards a known mechanism on a cellular level, but the entire complexity of physiological and pathophysiological processes can only be studied in vivo so far.
Dr. Jolkkonen: Apart from that, we need experimental models to investigate therapeutic 1) safety, 2) efficacy, and 3) potential influences of co-morbidities being relevant to stroke. This again is only possible in a living organism.
Dr. Zille: One important aspect for successful translation is target engagement, the evidence that a treatment reaches its therapeutic target. Biomarkers or surrogate measures, such as imaging, inflammatory, and other injury markers, need to be developed and validated in animal models and then used to assess injury and target engagement clinically.
Dr. Modo: The fundamental question remains if the brain is capable of repairing itself and if it lacks the environmental conditions to do so. Engineering strategies could, hence, be devised to overcome these environmental barriers. Animal models allow us to investigate what conditions could promote better tissue repair and eventually restore tissue that has been lost. It is currently presumptive to assume this will be producing an architecture akin to that which emerges during normal development. However, it is conceivable that different tissue architectures could support similar functional networks or support existing networks to work better. These might not produce a brain that is indistinguishable from a normal brain, but that is not the aim. Providing a new neural substrate to promote restoration of function is the key outcome we are looking for.
Dr. Shekhar: What are the challenges faced in implementing true tissue restoration?
Dr. Boltze: Brain tissue genesis in a large scale is only possible during the early stages of individual development. It requires the precise spatial and temporal orchestration and interaction of numerous cells, and even minor disturbances may dramatically affect this delicate process. Many biochemical and anatomical cues are required for brain tissue genesis. This situation is similar to other organs, but the cytoarchitecture of the brain is the most complex in the human body, and many of those cues do not exist in the adult brain anymore. Moreover, pathophysiological stroke consequences disturb the limited endogenous restoration capacities of the organ. This illustrates the complexity of the problem the field has to deal with.
Dr. Jolkkonen: It may be naïve to assume that transplanted cells do exactly what we expect them to do to repair the brain. It is even questionable whether the majority of transplanted cells remain functional. Nevertheless, tissue neoformation is possible as, for instance, illustrated by the turnover of oligodendrocyctes in the adult brain.
Dr. Modo: I see 3 key challenges: 1) Technically what, how and where should we inject something to invoke an appropriate tissue restoration response? 2) What will be the tissue response, and how do we adjust what, how and where we inject material? 3) How do we distinguish existing and new brain tissue from each other if these are well integrated?
Dr. Shekhar: You discussed four key strategies to improve the tissue repair and replacing in stroke. Could you go over it briefly?
Dr. Boltze: We suggest four research components. The first is to use restoration-permissive stroke models featuring comparatively small lesions or predominant affection of individual cell types. This preserves some of the anatomical and biochemical cues required to generate new tissue. If we were able to repair these smaller lesions, we may have a chance to successfully address the massive damage caused by larger territorial strokes.
Dr. Modo: Second, we suggest using functional biomaterials where possible to provide protection, stimulation and guidance of cell-based tissue restoration in situ. Advances in the biomaterials field have been tremendous in the recent years.
Dr. Jolkkonen: Third, we suggest the timely application of neurorehabilitation, which is known to rely on endogenous restoration capacities and neuroplasticity. This may support the externally delivered tools, and, ideally, both repair processes would work together.
Dr. Boltze: Finally, we suggest the use of supportive pharmacotherapy. Recent publications indicate that there is the possibility to pharmacologically control and augment brain plasticity. Future research may also pave the way to drug-based, “indirect” control of a cell graft.
Dr. Shekhar: In your paper, you mention large animal models. Why do you think they are important, in addition to studying rodent models?
Dr. Boltze: Large animal models cannot replace rodent stroke models. However, the human brain is among the largest of all mammalian species. Some elements of the potential brain repair strategies the field envisions, including but not limited to stem cell migration and paracrine effects, critically depend on time, distance and dimensions. Those can only be realistically modeled in larger brains.
Moreover, the anatomy of large animal brains is more similar to that of humans. Examples comprise the gyrencephalic cortex and the higher white matter content. Large animals are also perfectly suited for imaging-based therapy guidance and surveillance that is most likely to be employed in future human patients. We, therefore, think that large animal models are valuable for therapy validation and refinement in late-stage preclinical research.
Dr. Zille: Another example is hematoma resolution that takes much longer in humans as compared to rodents because of bigger absolute hematoma volumes, which can be modeled in larger animals. Large animal models also provide the opportunity to investigate tissue repair strategies following hematoma evacuation, especially combinatorial treatments involving biomaterials and pharmacologic agents.
Dr. Modo: A key challenge in regenerative medicine is vascularization. Smaller volumes are relatively easy to re-vascularize, especially using smaller blood vessels that support the parenchyma they are embedded in. In contrast, larger structures also need a large vascular supply that can “pump” sufficient volume to supply smaller vessels embedded in the parenchyma. Even rat models are not sufficiently large to design these types of strategies. Eventually, large gyrencephalic brain will, hence, be required to evaluate design strategies to replace large volumetric tissue defects.
Dr. Shekhar: What are the limitations in developing successful animal models?
Dr. Jolkkonen: A major problem is to study human cells in immunocompetent rodent models. Modeling stroke in immune-compromised mice is possible but challenging, and rodents with ‘humanized’ immune systems still have numerous limitations. In addition, rodents exhibit a much higher plasticity than humans, while spontaneous recovery may lead to overestimation of treatment effects.
Dr. Boltze: Even the best model is just a simplified copy of reality. Combining promising models may bring us a bit closer to reality, though.
Dr. Modo: Models are designed to test a particular set of circumstances thought to be relevant to the overall goal. Small rodent models serve some of the criteria to be fast and inexpensive to improve our understanding of the basic biology and requirement to define new therapeutic strategies. They can provide design guidance to move to larger and more complex models, such as non-human primates, or canine patients presenting in veterinary hospitals, where brain volume and immunology will be more important to evaluate interventions and refine these further. However, in contrast to rodent models, only small numbers of subjects will be used. These will essentially provide confirmation that additional design features were sufficiently addressed to move towards initial human safety trials.
Dr. Zille: Current animal models of stroke and other neurodegenerative diseases account for signs and symptoms that are already installed in afflicted patients. This is because they have been created in retrospect based on what it is already present in the patient’s ill brain. Hence, it is difficult to discern real cause from mere consequences with current animal models. Given the fact that the etiologic processes leading to stroke are likely life-spanning starting silently at least 20 to 30 years before the diagnosis is made, it is important to investigate these changes in animal models. However, use of spontaneous stroke models will likely require greater animal numbers and specialized equipment to detect the occurrence of stroke.
Dr. Shekhar: You have mentioned that the new treatment strategies may not be applied to every patient. How do you recommend overcoming this limitation?
Dr. Boltze: Indeed, we think that true tissue restoration is so complex and challenging that we have to start with the easy tasks, i.e. the repair of small and focused lesions. Once we have learnt how to successfully address those, we may scale up our tools to address the larger challenges. We need a stepwise development approach and much more time to learn the basics of true brain repair to maintain the chance to be ultimately successful.
Dr. Jolkkonen: Apart from that, careful selection of patients is needed. Some patients or lesion types/configurations may be more susceptible to supportive interventions than others. We need blood and imaging biomarkers to select the right patient for the right approach.
Dr. Modo: Patients present with very different types of tissue damage after a stroke. Some will have cavitation, where all tissue integrity is gone, whereas others will mostly have lost cells, but some tissue structure remains. Being able to distinguish patients based on their pathology will allow us to define which patients might benefit from implantation of only cells (in case tissue structure remains), versus those that might benefit from additional support, such as biomaterials plus cells to fill a tissue cavity. Non-invasive imaging will increasingly play a role to identify and stratify patients to appropriate treatments, but also be required to guide these advanced therapies to the right position in the stroke-damaged brain.
Dr. Zille: We need to understand the heterogeneity of the patient population and the mechanisms underlying the different types of stroke. For example, recent experimental evidence suggests that the pathophysiological mechanisms underlying ischemic and hemorrhagic stroke are different. Therefore, therapeutic strategies to promote tissue repair after ischemic and hemorrhagic stroke may, in part, be different.
Dr. Shekhar: The paper has a nice figure laying out the steps that are necessary to bring the successful results from the benchwork to the clinical use. Could you go through it briefly?
Dr. Boltze: The first central message of the figure is that clinically meaningful and well-defined scenarios should inform the in vivo research strategy to be employed. This shall make sure that the development made exactly fits the requirements in the particular clinical situation of interest. This automatically means that we restrict our research to well-defined target patient populations.
Second, the model best mimicking this population should be selected (research component 1), followed by an orchestrated implementation of the other research components. This leads to a first and basic efficacy screening in the model.
In case the approach was successful, the influence of important confounders and comorbidities is tested in the next step. Since we cannot investigate the influence of all possible confounders and comorbidities, we suggest focusing on those being most critical in the target patient population and to implement them in an advanced efficacy assessment experiment.
Finally, a thorough safety assessment may pave the way to early stage, safety-oriented clinical trials in the targeted patient population, while optional and parallel therapy optimization studies, also employing large animal models, prepares later, efficacy-focused studies.
Dr. Modo: It is important to recognize that models are designed for specific purpose and aligned with the question that is asked. As we recognize that different biological aspects are important to how a treatment works in stroke, we need to determine if other aspects influence this treatment. Different models, therefore, serve this purpose and improve our understanding of which patients will benefit most from these new interventions. To ensure that therapies find an efficient path to clinical implementation, it is important to ensure safety, but some of the finer details might not be required for testing in a well-defined cohort of patients. This does not mean to forget about the more rigorous pre-clinical studies, but it means progress can be achieved in both the clinical and pre-clinical arena simultaneously. Steady progress is guaranteed through exchange between both, rather than completing one before the other can start.
Dr. Shekhar: What recommendations do you have for the new generations of clinical or non-clinical researchers who want to develop these treatment strategies?
Dr. Jolkkonen: We should be critical with our results and those found in the literature. The relative lack of reports on neutral or negative (experimental) data has caused too optimistic expectations and overpromises in the past. We should instead focus on thorough and stepwise, but continuous, research programs.
Dr. Zille: We also believe that a thorough research strategy requires collaboration between different labs being specialized in particular aspects to develop a particular treatment strategy — as suggested by a number of excellent publications from colleagues in our field.
Dr. Modo: We have to thread a careful path between being excited about new opportunities and not overpromising on their delivery. A careful study with reporting of all results is required to provide a balanced view and refine our questions. Hype can lead to overzealous expectations and introduce bias in our evaluations, especially if we lack experience in analyzing new phenomena, such as brain tissue restoration. It is also important to ensure long-term funding that supports the transition from the lab to the bench, and back. Failure is part of the system. Many therapies might not work on the first translation to the clinic, bringing it back to the lab, and refining the question could, nevertheless, provide stepwise advances, as seen in other fields of study. Funding agencies and university hospitals need to develop structures that can ensure this innovation process; otherwise, many promising therapies might take a long time to benefit patients.
Dr. Boltze: I could not agree more. We all should be modest in our expectations and patient when trying to realize them. We also should carefully analyze past failures to really learn from them. As Dr. Jolkkonen mentioned, a constructive discussion of negative results should be part of that. When embarking on clinical trials, we should avoid addressing optimistic efficacy endpoints in early stage trials that are simply not powered to investigate those. We may miss a number of smaller, but nevertheless meaningful, effects and prematurely exit from promising therapeutic avenues.n