Developmental Regulation of Matrix Metalloproteinases in Response to Multifactorial, Severe Traumatic Brain Injuries during Immaturity

Abstract

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 PND7 or PND30 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 h 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 MMPs 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.

Keywords: Animal model; Blood-brain barrier; Developing brain; Hypoxic-ischemic injury; Inflammation.

Fig. 1.

AHT model injuries caused widespread albumin extravasation in the ipsilateral hemisphere in PND30 pigs and minimal albumin extravasation in PND7 piglets. Albumin extravasation could be observed macroscopically in piglets with HH model injuries (albumin = brown, healthy tissue = purple; b) versus sham pigs (healthy tissue = purple; lines = 1 mm increments; a). Due to the potential for artifact, distribution was mapped by albumin extravasation microscopically. c Photomicrograph of healthy cortex where albumin is contained to the blood vessels (scale bar = 100 μM). d Photomicrograph of damaged cortex where albumin is widespread (scale bar = 100 μM). e A representative, single-section ipsilateral versus contralateral to focal injuries in representative piglets receiving model injuries or sham surgery demonstrating that the pattern of albumin extravasation (purple) is similar to the pattern of damage seen via H&E (yellow). f Percentage of the hemisphere with albumin extravasation ipsilateral or contralateral to focal injuries in PND7 and PND30. ^a,bMeans ± SD with different letters differ (p < 0.05).

Fig. 2.

MMP-9 upregulation was increased in PND30 but not PND7 after AHT model injuries. a Minimal matrix metalloproteinase-9 (MMP-9) expression in healthy cortex (scale bar = 50 μM). MMP-9 was upregulated in neurons (scale bar = 50 μM, b; scale bar = 20 μM, d), glia (scale bar = 50 μM, c), and the basement membrane of blood vessels (scale bar = 20 μM, e) in damaged tissue. Though the interstitial matrix was often positive for MMP-9, areas were only mapped as positive if cells in that area were also positive. f Areas of albumin extravasation (purple) and increased MMP-9 expression (blue) were in a representative mapped section in injured and sham piglets. g The percentage of the hemisphere with MMP-9 upregulation was greater in the hemisphere ipsilateral to focal injuries compared to the contralateral hemisphere in PND30 but equivalent among hemispheres in PND7. h In sections with over 30% damage, damage areas via H&E (yellow) were equivalent, but PND30 had greater areas of vasogenic edema as detected via albumin extravasation compared to PND7. Albumin extravasation was less than the histologic tissue damage in PND7. Both these characteristics might indicate that the pathophysiology may be continuing to evolve in PND30 while spreading has ceased in PND7 piglets.

Fig. 3.

Active MMP-3 and MMP-9 is upregulated in PND30 but not PND7 piglets in response to AHT model injuries. a Western blot of matrix metalloproteinase-3 and -9 (MMP-3, -9), actin, and Ponceau total protein stain in the rostral gyrus rostral or lateral from the cortical impact or sham surgery of PND7 and PND30 piglets at injury 1 or 24 h after injury or sham injury (samples run in duplicate). b MMP-3 proenzyme did not increase in either age in response to injury, while active MMP-3 increased in PND30 piglets but not in PND7 piglets. c Pro-MMP-9 did not increase in either age, but active MMP-9 was greater at 1 h post-injury in PND30 versus PND7 piglets. Differences among groups were compared via two-way ANOVA, followed by Tukey’s post hoc tests, and p ≤ 0.06 was considered significant. d Western blot of albumin, actin, and Ponceau stain in the rostral gyrus rostral or lateral from the cortical impact or sham surgery of PND7 and PND30 piglets at injury 1 or 24 h after injury or sham injury (samples run in duplicate). e Albumin was increased in PND30 piglets after AHT model injuries.

Fig. 4.

Interaction of inflammatory mediators and seizure. a Seizure duration was longer in piglets surviving 24 h versus 1 h, but there was no effect of age on seizure duration. Seizure duration was negatively correlated to total MMP-9 and pro-MMP-9 (b) but was not correlated with active MMP-9, pro-MMP-3, active MMP-3, nor albumin (c) (Pearson correlations). d Table of the correlation and p values for all mediators.

Fig. 5.

The activity of MMP-2 and -9 in peripheral plasma in response to AHT model injuries is developmentally regulated. Gelatin zymography with matrix metalloproteinase-9 (MMP-9) standard and the pro- and active forms of MMP-2 and -9 in one PND7 (a) and two PND30 (b) piglets over time. c Pro-MMP-9 increased in both ages (MMP activity is expressed as a ratio to pre-injury). d Active MMP-9 only increased in PND30 (p = 0.01). Pro-MMP-2 (e) and active MMP-2 (f) only increased in PND30 piglets. Samples are displayed a ratio to pre-injury. ^a,b,cMeans ± SD with different letters differ p < 0.05.