Supplementary Material for: Developmental regulation of matrix metalloproteinases in response to multi-factorial, severe TBI injuries during immaturity.

Introduction: A striking pattern in young children after severe TBI is when the entire cortical ribbon displays tissue damage: hemispheric hypodensity (HH). HH is often a result of abusive head trauma (AHT). We previously reported a model of HH in a gyrencephalic species where a combination of injuries consisting of 1) cortical impact, 2) midline shift, 3) subdural hematoma/subarachnoid hemorrhage, 4) traumatic seizures, and 5) brief apnea and hypoventilation, resulted in extensive, hypoxic-ischemic type injury. Importantly, this mechanism closely resembles that seen in children, with relative sparing of the contralateral cortex, thus, ruling out a pure asphyxia mechanism. In this model, piglets of similar developmental stage to human toddlers (postnatal day 30, PND30) have extensive hypoxic-ischemic damage to the cortical ribbon with sparing of the contralateral hemisphere and deep gray matter areas. However, piglets of similar developmental stage to human infants (postnatal day 7, PND7) have less hypoxic-ischemic damage that is notably bilateral and patchy. We therefore sought to discover whether the extensive tissue damage observed in PND30 was due to a greater upregulation of matrix metalloproteinases (MMPs).
Materials and Methods: In PND 7 or PND 30 piglets receiving AHT injuries (cortical impact, midline shift, subdural hematoma/subarachnoid hemorrhage, traumatic seizures, and brief apnea and hypoventilation) or a sham injury, the pattern of albumin extravasation and MMP-9 upregulation throughout the brain was determined via immunohistochemistry, brain tissue adjacent to the cortical impact where the tissue damage spreads was collected for Western blots, and the gelatinase activity was determined over time in peripheral plasma. EEG was recorded and piglets survived up to 24 hours after injury administration.
Results: The pattern of albumin extravasation, indicating vasogenic edema, as well as increase in MMP-9, were both present at the same areas of hypoxic-ischemic tissue damage. Evidence from immunohistochemistry, western blot, and zymogens demonstrate that MMP- 2,- 3 or -9 are constitutively expressed during immaturity and are not different between developmental stages; however, active forms are upregulated in PND30 but not PND7 after in response to AHT model injuries. Furthermore, peripheral active MMP-9 was downregulated after model injuries in PND7.
Conclusions: This differential response to AHT model injuries might confer protection to the PND7 brain. Additionally, we find that immature gyrencephalic species have a greater baseline and array of MMP’s than previously demonstrated in rodent species. Treatment with an oral or intravenous broad-spectrum matrix metalloproteinase inhibitor might reduce the extensive spread of injury in PND30, but the exposure to metalloproteinase inhibitors must be acute as to not interfere with the homeostatic role of matrix metalloproteinases in normal postnatal brain development and plasticity as well as post-injury synaptogenesis and tissue repair.

引言：重度创伤性脑损伤（Traumatic Brain Injury, TBI）后幼儿的典型表现为全皮层脑回出现组织损伤，即半球低密度（hemispheric hypodensity, HH）。HH常由虐待性头部创伤（abusive head trauma, AHT）引发。我们此前曾在沟回型脑物种（gyrencephalic species）中建立HH模型，通过联合施加5类损伤：1）皮层撞击损伤（cortical impact）、2）中线移位（midline shift）、3）硬膜下血肿/蛛网膜下腔出血（subdural hematoma/subarachnoid hemorrhage）、4）创伤性癫痫（traumatic seizures）、5）短暂呼吸暂停与低通气（brief apnea and hypoventilation），最终诱导出广泛的缺氧缺血性损伤（hypoxic-ischemic type injury）。值得注意的是，该损伤机制与儿童中的发病机制高度相似，且对侧皮层（contralateral cortex）相对保留未受损伤，从而排除了单纯窒息性损伤的机制。在该模型中，发育阶段与人类学步儿童相当的仔猪（出生后第30天，postnatal day 30, PND30）会出现广泛的皮层脑回缺氧缺血性损伤，而对侧半球及深部灰质区域则得以保全。然而，发育阶段与人类婴儿相当的仔猪（出生后第7天，postnatal day 7, PND7）的缺氧缺血性损伤程度更轻，且呈现显著的双侧性、斑片状分布。因此，我们试图探究PND30仔猪中观察到的广泛组织损伤是否由基质金属蛋白酶（matrix metalloproteinases, MMPs）的更高水平上调所介导。

材料与方法：对出生后第7天（postnatal day 7, PND7）或出生后第30天（postnatal day 30, PND30）的仔猪施加AHT损伤（包括皮层撞击损伤、中线移位、硬膜下血肿/蛛网膜下腔出血、创伤性癫痫及短暂呼吸暂停与低通气）或假损伤，通过免疫组织化学（immunohistochemistry）法检测全脑范围内白蛋白外渗（albumin extravasation）与基质金属蛋白酶-9（MMP-9）上调的模式；收集皮层撞击损伤周边的损伤扩散波及脑组织进行蛋白质印迹（Western blots）检测；并在不同时间点检测外周血浆（peripheral plasma）中的明胶酶活性（gelatinase activity）。同时记录脑电图（electroencephalogram, EEG），仔猪在损伤造模后可存活长达24小时。

结果：白蛋白外渗（提示血管源性水肿（vasogenic edema））以及MMP-9水平升高的分布模式，均与缺氧缺血性组织损伤的区域一致。免疫组织化学、蛋白质印迹及酶原（zymogens）检测结果显示，MMP-2、-3或-9在发育未成熟阶段呈组成型表达，且在不同发育阶段的仔猪间无显著差异；但在AHT模型损伤后，PND30仔猪的MMP活性形式（active forms）出现上调，而PND7仔猪则无此变化。此外，PND7仔猪在造模损伤后，外周血中的活性MMP-9水平出现下调。

结论：这种对AHT模型损伤的差异性反应，可能为PND7仔猪的大脑提供了保护作用。此外，我们发现沟回型脑未成熟物种的基质金属蛋白酶（MMPs）基线水平及种类均高于此前在啮齿类物种中的报道。口服或静脉给予广谱基质金属蛋白酶抑制剂（broad-spectrum matrix metalloproteinase inhibitor），或可减轻PND30仔猪的损伤广泛扩散，但需采用急性给药方案，以免干扰基质金属蛋白酶在正常出生后脑发育与可塑性（postnatal brain development and plasticity）、以及损伤后突触发生与组织修复（post-injury synaptogenesis and tissue repair）中的稳态（homeostatic role）作用。