Mathematical measures of complexity shed light on why some concepts are inherently more difficult to learn than others

“It was not only difficult for him to understand that the generic term dog embraced so many unlike specimens of differing sizes and different forms; he was disturbed by the fact that a dog at three-fourteen (seen in profile) should have the same name as the dog at three-fifteen (seen from the front).…Without effort, he had learned English, French, Portuguese, Latin. I suspect, nevertheless, that he was not very capable of thought. To think is to forget a difference, to generalize, to abstract. In the overly replete world of Funes there were nothing but details, almost contiguous details.” —Jorge Luis Borges, “Funes the Memorius”“It was not only difficult for him to understand that the generic term dog embraced so many unlike specimens of differing sizes and different forms; he was disturbed by the fact that a dog at three-fourteen (seen in profile) should have the same name as the dog at three-fifteen (seen from the front).…Without effort, he had learned English, French, Portuguese, Latin. I suspect, nevertheless, that he was not very capable of thought. To think is to forget a difference, to generalize, to abstract. In the overly replete world of Funes there were nothing but details, almost contiguous details.” —Jorge Luis Borges, “Funes the Memorius”“It was not only difficult for him to understand that the generic term dog embraced so many unlike specimens of differing sizes and different forms; he was disturbed by the fact that a dog at three-fourteen (seen in profile) should have the same name as the dog at three-fifteen (seen from the front).…Without effort, he had learned English, French, Portuguese, Latin. I suspect, nevertheless, that he was not very capable of thought. To think is to forget a difference, to generalize, to abstract. In the overly replete world of Funes there were nothing but details, almost contiguous details.” —Jorge Luis Borges, “Funes the Memorius”We are told scientists are divided into experimentalists and theoreticians. The dialectic description of the dynamics of science, with one tribe gathering data and collecting evidence and another tribe providing form to these observations, has striking examples that argue for the importance of synthesis. The 16th century revolution, which settled the way in which we see the sky today, is probably one of the best examples of how comparatively ineffective each of these tribes can be in isolation. Tycho Brahe, the exquisite observer, who built, calibrated, and refined instruments to see in the sky what no one else could, collected the evidence to prove a theory that Copernicus had already stated years before (in a book he dedicated to the Pope). It was only many years later that Galileo established the bridge between theory and observation; he understood the data in terms of the theory and thereby cemented the revolution. Copernicus's statements, showed Galileo, were not only figments of his imagination; they were an adequate description of the universe as he and Brahe had observed it.Since my first steps in biology, after a prompt departure from physics and mathematics, I have looked for such encounters between theory and experiment. I began studying the visual system and the series of fundamental works by  Atneave (1954) ,  Barlow (1960) , and  Atick (1992)  on the relationship between our visual world and the way the brain processes it. Their research was based on a simple hypothesis: (1) the images that we see are highly redundant and (2) the optic nerve is a limited channel, thus the retina has to compress information. Compression, redundancy? How do such concepts relate to the biology of the brain?In the middle of the last century, working on the problem of communications, languages, codes, and channels, Claude Shannon proposed a very elegant theory that formalized intuitive ideas on the essence (and the limits) of communications ( Weaver and Shannon 1949 ). One of its key aspects was that, depending on the code and on the input, channels are not used optimally and thus do not carry all the information they potentially could. When we compress (zip) a file, we are actually rewriting it in a more efficient (though not necessarily more intelligible) language in which we can store the same amount of information in less space. This compression has, of course, a limit (we cannot convey the universe in a single point), and the mere existence of an optimal code is central to Shannon's idea. Attneave was probably the first to think that these ideas could help unravel how the brain worked, and in a long series of works relating these ideas to experimental data, it was shown that the retina's business is mainly to get rid of the redundancies in the visual world.About four years ago, Jacob Feldman published a paper, similar in spirit, proposing a simple explanation for a long-standing problem in cognitive science about why some concepts are inherently more difficult to learn than others ( Feldman 2000 ). An article whose first reference is to work carried out 50 years previously makes us suspect that an important gap is being filled. As in the previous experiments, Feldman borrowed a theory—he did not invent it—to explain longstanding and previously unexplained research in this area. Feldman's idea was based on a theory developed by Kolmogorov that established formal mathematical grounds to define and measure complexity. Kolmogorov's theory was focused on categories, which are just subsets of a universe, a bunch of exemplars that constitute a group. “Dogs” is a category in the universe of animals. Different statements can define the same category, for example, “big dogs” and “dogs that are not small” are the same group, and some information in the statement may be irrelevant because it does not aid in determining which elements belong or not. In the same way that Shannon spoke of a non-redundant code, Kolmogorov showed that categories could be defined with an optimal (non-redundant) statement. The length of this statement defines a measure of complexity termed Kolmogorov complexity.To visualize the intuitive nature of his theory it helps to do a thought experiment. Imagine a set of objects defined by, say, three features: form, color, and size. And imagine, for simplicity, that each feature is binary, that is, there are only two possible cases. Size is big or small, color is yellow or red, and shape can be either a triangle or a circle. This defines, of course, a universe of eight objects. We can now define categories within this universe: for example, all the circles (a category of four exemplars), or all the big and yellow objects (two exemplars), or all the triangles that are not red (again two) (see Figure 1). We can also define a category by enumeration, for example, the small red triangle, the big yellow circle, and the small yellow circle (three exemplars). Some rules (and thus the groups defined by these rules) are intuitively easier to define than others. “All the circles” is an easier statement to make (and probably to remember) than “small circles and yellow big objects.” This notion of difficulty is what Kolmogorov's theory formalized, stating that complexity was the length of the shortest definition (among all the possible definitions) of a given set. From this thought experiment, we can understand the logic of Feldman's paper, which showed that. Kolmogrov complexity is very closely related to our intuitive notion of conceptual difficulty. Feldman presented subjects with all possible categories (of a fixed number of exemplars) in different universes and showed that the critical parameter to rank the difficulty of a given subset was its Kolmogrov complexity. Moreover by explicitly presenting all the members and the nonmembers of a category to naïve subjects, he showed that we can spontaneously reduce a category to its minimal form and remember it without any explicit instruction. Thus, what Feldman found, following the original ideas of Shepard, was that our psychological measure of complexity—our difficulty in defining and remembering a category or concept—is also determined by the Kolmogorov complexity that describes it.This essay is, in a way, about how we avoid becoming Borges's character Funes, who could not understand repeated observations as exemplars of a common rule and thus could not synthesize and categorize. Simply, he could not think. Probably the most disappointing moment of Feldman's paper comes at the very end, where it deals with its (somehow unavoidable) recursive quest. Understanding why some concepts are difficult to learn may itself be difficult to learn. Modern mathematics, together with Kolmogorov complexity and information theory, has taught us another fundamental concept that may be relevant when trying to understand the logic of the mind. In a long series of paradoxes enumerated by Bertrand Russell, Kurt Goedel, and others, we learn that a formal system that looks at itself is bound to fail. At the very end of his paper, Feldman writes, “In a sense, this final conclusion [that psychological complexity is Boolean complexity] may seem negative: human conceptual difficulty reflects intrinsic mathematical complexity after all, rather than some idiosyncratic and uniquely human bias.” Who invented mathematics? The Martians? On the contrary, I believe this result supports a more naturalistic and less Platonic conception of mathematics. Formal ideas in mathematics are not arbitrary constructions of an arbitrary architecture; rather, they reflect the workings of the brain like a massive collective cognitive experiment. Mathematics does not only serve to help us understand biology; mathematics is biology. We are not less original if our thoughts resemble our mental constructions, we are just consistent. It is within this loop, this unavoidable recursion—mathematics understanding the logic of the brain—that we will have an opportunity to test, as some conspire, whether among all the wonders evolution has come out with, the ultimate might be a brain good enough to avoid the risk of understanding itself.


To quickly control infectious disease outbreaks, extensive information is required to identify the source and transmission routes, and to evaluate the effect of containment policies. Traditionally, scientists have used travel- and contact-tracing methods, but the recent SARS epidemic showed that sequence-based techniques for pathogen detection can also be important tools to help understand outbreaks. Jianjun Liu and colleagues adapted mass spectrometry (MS)–based genotyping, already used as a high-throughput way of detecting single nucleotide polymorphisms in human DNA, to the analysis of the SARS virus from clinical samples.The major breakthroughs against SARS were the discovery of the SARS coronavirus (SARS-CoV) as the etiological agent and the sequencing of the SARS genome. Liu's colleagues at the Genome Institute of Singapore had previously shown that common genetic variants in the SARS-CoV genome could be used as molecular fingerprints to help trace the route of infection. However, as “sequence analysis of large numbers of clinical samples is challenging, cumbersome, and expensive,” they felt that “what is needed is a rapid, sensitive, high throughput, and cost-effective screening method.” Towards this goal, Liu and colleagues now demonstrate that an MS-based technique can quickly yield accurate information on clinical isolates (in this case from the 2003 SARS outbreak in Singapore).The scientists demonstrate the sensitivity of the assay in detecting SARS-CoV variations and test it further in cultured viral isolates and uncultured lung tissue samples of SARS-CoV. They analyzed isolates taken from 13 patients with SARS at different stages of the Singapore outbreak, identified nine sequence variations, and discovered a new primary route of introduction of the virus into the Singapore population. They also found a Singaporean origin for a German case of SARS, a result that could not be derived from standard sequencing methods. The analysis of the uncultured lung tissue also found different sequences in a single patient, which suggested the presence of multiple viral sequence variants in one host.The study suggests that MS-based genotyping can be used for large-scale genetic characterization of viral DNA from clinical samples. The researchers found that the method was accurate and sensitive, with a 95% success rat e for detecting sequence variations at low virus concentrations. The MS-based assay allows high-throughput analysis and complements the “gold standard” direct sequence analysis method, which is used to identify new sequence variations. As such, it is particularly useful for investigating agents for which extensive sequence information exists.Liu and colleagues propose that the most efficient method for a large-scale population investigation would be initial characterization of a genome sequence by direct sequence analysis in a subset of samples, followed by MS-based analysis of informative genetic variations. Altogether, their results suggest that MS-based genetic analysis can help real-time investigations in disease outbreaks.


Background The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline to suppress matrix metalloproteinase-9 (MMP-9) in brain arteriovenous malformations (AVMs).
Background
The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline to suppress matrix metalloproteinase-9 (MMP-9) in brain arteriovenous malformations (AVMs).Methods Ex-vivo treatment of AVM tissues:  Intact AVM tissues were treated with doxycycline for 48 hours. Active and total MMP-9 in the medium were measured.  Pilot trial:  AVM patients received either doxycycline (100 mg) or placebo twice a day for one week prior to AVM resection. Active and total MMP-9 in BVM tissues were measured.
Methods
Ex-vivo treatment of AVM tissues:  Intact AVM tissues were treated with doxycycline for 48 hours. Active and total MMP-9 in the medium were measured.  Pilot trial:  AVM patients received either doxycycline (100 mg) or placebo twice a day for one week prior to AVM resection. Active and total MMP-9 in BVM tissues were measured.Ex-vivo treatment of AVM tissues:Pilot trial:Results Ex-vivo treatment of AVM tissues:  Doxycycline at 10 and 100 μg/ml significantly decreased MMP-9 levels in AVM tissues ex-vivo (total: control vs 10 vs 100 μg/ml = 100 ± 6 vs 60 ± 16 vs 61 ± 9%; active: 100 ± 8 vs 48 ± 16 vs 59 ± 10%).  Pilot trial:  10 patients received doxycycline, and 4 patients received placebo. There was a trend for both MMP-9 levels to be lower in the doxycycline group than in the placebo group (total: 2.18 ± 1.94 vs 3.26 ± 3.58, P = .50; active: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25).
Results
Ex-vivo treatment of AVM tissues:  Doxycycline at 10 and 100 μg/ml significantly decreased MMP-9 levels in AVM tissues ex-vivo (total: control vs 10 vs 100 μg/ml = 100 ± 6 vs 60 ± 16 vs 61 ± 9%; active: 100 ± 8 vs 48 ± 16 vs 59 ± 10%).  Pilot trial:  10 patients received doxycycline, and 4 patients received placebo. There was a trend for both MMP-9 levels to be lower in the doxycycline group than in the placebo group (total: 2.18 ± 1.94 vs 3.26 ± 3.58, P = .50; active: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25).Ex-vivo treatment of AVM tissues:Pilot trial:Conclusions A clinically relevant concentration of doxycycline decreased MMP-9 in ex-vivo AVM tissues. Furthermore, there was a trend that oral doxycycline for as short as one week resulted in a decrease in MMP-9 in AVM tissues. Further studies are warranted to justify a clinical trial to test effects of doxycycline on MMP-9 expression in AVM tissues.
Conclusions
A clinically relevant concentration of doxycycline decreased MMP-9 in ex-vivo AVM tissues. Furthermore, there was a trend that oral doxycycline for as short as one week resulted in a decrease in MMP-9 in AVM tissues. Further studies are warranted to justify a clinical trial to test effects of doxycycline on MMP-9 expression in AVM tissues.

Background Brain arteriovenous malformations (AVM) represent a relatively infrequent but devastating source of neurological morbidity in relatively young adults [ 1 ]. Prevention of new or recurrent intracranial hemorrhage (ICH) is the primary rationale for treating AVMs. The optimal management of AVMs is not well defined, and the risk of aggressive surgical therapy can be significantly high. There is a subset of AVM patients that are considered to be inoperable due to the location and size of their lesions [ 2 ]. To date, there is no clinically available pharmacological treatment of inoperable AVMs to decrease the rate of spontaneous intracranial hemorrhage. Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, degrade extracellular matrix proteins, cell surface molecules, and other peri-cellular substances [ 3 ]. Excessive degradation of the vascular matrix by MMPs may result in the destabilization of the blood vessel that potentially leads to weakening of the vessel wall, passive dilatation, and rupture [ 4 ]. Previously, we reported increased levels of MMP-9 activity in AVMs that may result in vascular instability associated with growth and bleeding. There is an increasing interest in utilizing MMP inhibitors in treating vascular diseases including abdominal aortic aneurysms. Doxycycline is a clinically available antibiotic agent that possesses non-specific inhibitory effects on various MMPs, and for years it has had a well-established safety record in treating infectious diseases. The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline as an MMP inhibitor to decrease MMP-9 activity in AVMs and potentially decrease the rate of spontaneous hemorrhage. This exploratory investigation supports the concept that further studies be conducted to document the ability of tetracycline and its derivatives to decrease MMP-9 levels in AVM nidal tissue. Such a demonstration could provide a firm rationale for proceeding with clinical trials to test the hypothesis that tetracycline and its derivatives are useful to decrease the rate of spontaneous hemorrhage from AVMs in otherwise untreatable patients or in those awaiting interventional treatment. First, we demonstrate that doxycycline can decrease MMP-9 activity in cultured AVM tissues as a proof of the concept. Further, we present results from a pilot clinical study demonstrating effects of oral doxycycline treatment on MMP-9 expression in AVM tissues.
Background
Brain arteriovenous malformations (AVM) represent a relatively infrequent but devastating source of neurological morbidity in relatively young adults [ 1 ]. Prevention of new or recurrent intracranial hemorrhage (ICH) is the primary rationale for treating AVMs. The optimal management of AVMs is not well defined, and the risk of aggressive surgical therapy can be significantly high. There is a subset of AVM patients that are considered to be inoperable due to the location and size of their lesions [ 2 ]. To date, there is no clinically available pharmacological treatment of inoperable AVMs to decrease the rate of spontaneous intracranial hemorrhage.Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, degrade extracellular matrix proteins, cell surface molecules, and other peri-cellular substances [ 3 ]. Excessive degradation of the vascular matrix by MMPs may result in the destabilization of the blood vessel that potentially leads to weakening of the vessel wall, passive dilatation, and rupture [ 4 ]. Previously, we reported increased levels of MMP-9 activity in AVMs that may result in vascular instability associated with growth and bleeding.There is an increasing interest in utilizing MMP inhibitors in treating vascular diseases including abdominal aortic aneurysms. Doxycycline is a clinically available antibiotic agent that possesses non-specific inhibitory effects on various MMPs, and for years it has had a well-established safety record in treating infectious diseases.The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline as an MMP inhibitor to decrease MMP-9 activity in AVMs and potentially decrease the rate of spontaneous hemorrhage. This exploratory investigation supports the concept that further studies be conducted to document the ability of tetracycline and its derivatives to decrease MMP-9 levels in AVM nidal tissue. Such a demonstration could provide a firm rationale for proceeding with clinical trials to test the hypothesis that tetracycline and its derivatives are useful to decrease the rate of spontaneous hemorrhage from AVMs in otherwise untreatable patients or in those awaiting interventional treatment.First, we demonstrate that doxycycline can decrease MMP-9 activity in cultured AVM tissues as a proof of the concept. Further, we present results from a pilot clinical study demonstrating effects of oral doxycycline treatment on MMP-9 expression in AVM tissues.Methods Ex-vivo treatment of cultured AVM tissues After institutional review and informed consent, we obtained AVM specimens after microsurgical resection. AVM nidus was dissected away from any adjacent brain tissue in the operating room and a representative portion of nidus tissue was used for ex-vivo treatment. Ex-vivo culture of AVM tissue was performed using previously described method with modifications [ 5 ]. AVM tissues were minced into 1–2 mm fragments and vigorously washed with phosphate buffered saline (PBS). Blood cells dissociated from the tissues were removed by filtration. AVM tissue fragments were placed onto cell culture inserts and immersed in cell culture medium. During the first 24 hours, AVM tissues were incubated in DME H-21 medium containing 10% fetal bovine serum (FBS) to aid in tissue recovery. Following this recovery period, tissue debris and dissociated cells were removed, and AVM tissues were incubated in DME H-21 medium containing 1% FBS for 4 hours. The tissue fragments were equally divided into 12–24 wells. Then AVM tissues were incubated in the medium containing PBS (control) or doxycycline (1, 5, 10 and 100 μg/ml). Each group included 3–5 wells. Size of the tissues dictated a number of treatment groups in each specimen. Medium from each well was collected after 48 hours. Active and total MMP-9 were measured using substrate zymography. Medium was mixed with SDS sample buffer (Invitrogen, Carlsbad, CA, U.S.A.) and separated under non-reducing conditions in a 10% zymogram gel (Invitrogen) containing 0.1% gelatin incorporated as a substrate. Recombinant MMP-2 and MMP-9 proteins (R&D systems) were used as positive controls. After running, the gel was incubated with renaturing buffer (Invitrogen). The gel was then incubated with developing buffer (Invitrogen) overnight at 37°C. The gel was then stained with colloidal blue stain (Invitrogen). Proteolytic bands in the zymogram gels were quantified using Image J Software (NIH). Tissue viability was assessed by measuring the amount of LDH (lactate dehydrogenase) released in the medium according to the manufacturer's instructions (Roche, Penzburg, Germany). Some of the tissue fragments were embedded in paraffin for histological assessment. Pilot clinical study Fourteen AVM patients received either 100 mg of doxycycline or placebo twice a day for one week, prior to elective AVM resection. During the AVM resection, AVM tissues were collected, and nidus tissues were frozen in liquid nitrogen. Frozen tissues were stored at -80°C until analysis. Clinical data were collected as previously described [ 6 ]. The specimens were homogenized and insoluble materials were removed by centrifugation at 3000 rpm for 5 minutes. We used total MMP-9 ELISA kit (R&D) and active MMP-9 ELISA kit (Amersham). Statistical analysis Data are presented as mean ± standard deviation. The data for total MMP-9 and active MMP-9 from ex-vivo AVM tissues are presented as a relative expression with control brain samples as 100%. We used ANOVA for comparison, and statistical significance was taken at  P  < .05.
Methods
Ex-vivo treatment of cultured AVM tissues After institutional review and informed consent, we obtained AVM specimens after microsurgical resection. AVM nidus was dissected away from any adjacent brain tissue in the operating room and a representative portion of nidus tissue was used for ex-vivo treatment. Ex-vivo culture of AVM tissue was performed using previously described method with modifications [ 5 ]. AVM tissues were minced into 1–2 mm fragments and vigorously washed with phosphate buffered saline (PBS). Blood cells dissociated from the tissues were removed by filtration. AVM tissue fragments were placed onto cell culture inserts and immersed in cell culture medium. During the first 24 hours, AVM tissues were incubated in DME H-21 medium containing 10% fetal bovine serum (FBS) to aid in tissue recovery. Following this recovery period, tissue debris and dissociated cells were removed, and AVM tissues were incubated in DME H-21 medium containing 1% FBS for 4 hours. The tissue fragments were equally divided into 12–24 wells. Then AVM tissues were incubated in the medium containing PBS (control) or doxycycline (1, 5, 10 and 100 μg/ml). Each group included 3–5 wells. Size of the tissues dictated a number of treatment groups in each specimen. Medium from each well was collected after 48 hours. Active and total MMP-9 were measured using substrate zymography. Medium was mixed with SDS sample buffer (Invitrogen, Carlsbad, CA, U.S.A.) and separated under non-reducing conditions in a 10% zymogram gel (Invitrogen) containing 0.1% gelatin incorporated as a substrate. Recombinant MMP-2 and MMP-9 proteins (R&D systems) were used as positive controls. After running, the gel was incubated with renaturing buffer (Invitrogen). The gel was then incubated with developing buffer (Invitrogen) overnight at 37°C. The gel was then stained with colloidal blue stain (Invitrogen). Proteolytic bands in the zymogram gels were quantified using Image J Software (NIH). Tissue viability was assessed by measuring the amount of LDH (lactate dehydrogenase) released in the medium according to the manufacturer's instructions (Roche, Penzburg, Germany). Some of the tissue fragments were embedded in paraffin for histological assessment.
Ex-vivo treatment of cultured AVM tissues
After institutional review and informed consent, we obtained AVM specimens after microsurgical resection. AVM nidus was dissected away from any adjacent brain tissue in the operating room and a representative portion of nidus tissue was used for ex-vivo treatment. Ex-vivo culture of AVM tissue was performed using previously described method with modifications [ 5 ].AVM tissues were minced into 1–2 mm fragments and vigorously washed with phosphate buffered saline (PBS). Blood cells dissociated from the tissues were removed by filtration. AVM tissue fragments were placed onto cell culture inserts and immersed in cell culture medium. During the first 24 hours, AVM tissues were incubated in DME H-21 medium containing 10% fetal bovine serum (FBS) to aid in tissue recovery. Following this recovery period, tissue debris and dissociated cells were removed, and AVM tissues were incubated in DME H-21 medium containing 1% FBS for 4 hours. The tissue fragments were equally divided into 12–24 wells. Then AVM tissues were incubated in the medium containing PBS (control) or doxycycline (1, 5, 10 and 100 μg/ml). Each group included 3–5 wells. Size of the tissues dictated a number of treatment groups in each specimen.Medium from each well was collected after 48 hours. Active and total MMP-9 were measured using substrate zymography. Medium was mixed with SDS sample buffer (Invitrogen, Carlsbad, CA, U.S.A.) and separated under non-reducing conditions in a 10% zymogram gel (Invitrogen) containing 0.1% gelatin incorporated as a substrate. Recombinant MMP-2 and MMP-9 proteins (R&D systems) were used as positive controls. After running, the gel was incubated with renaturing buffer (Invitrogen). The gel was then incubated with developing buffer (Invitrogen) overnight at 37°C. The gel was then stained with colloidal blue stain (Invitrogen). Proteolytic bands in the zymogram gels were quantified using Image J Software (NIH).Tissue viability was assessed by measuring the amount of LDH (lactate dehydrogenase) released in the medium according to the manufacturer's instructions (Roche, Penzburg, Germany). Some of the tissue fragments were embedded in paraffin for histological assessment.Pilot clinical study Fourteen AVM patients received either 100 mg of doxycycline or placebo twice a day for one week, prior to elective AVM resection. During the AVM resection, AVM tissues were collected, and nidus tissues were frozen in liquid nitrogen. Frozen tissues were stored at -80°C until analysis. Clinical data were collected as previously described [ 6 ]. The specimens were homogenized and insoluble materials were removed by centrifugation at 3000 rpm for 5 minutes. We used total MMP-9 ELISA kit (R&D) and active MMP-9 ELISA kit (Amersham).
Pilot clinical study
Fourteen AVM patients received either 100 mg of doxycycline or placebo twice a day for one week, prior to elective AVM resection. During the AVM resection, AVM tissues were collected, and nidus tissues were frozen in liquid nitrogen. Frozen tissues were stored at -80°C until analysis. Clinical data were collected as previously described [ 6 ].The specimens were homogenized and insoluble materials were removed by centrifugation at 3000 rpm for 5 minutes. We used total MMP-9 ELISA kit (R&D) and active MMP-9 ELISA kit (Amersham).Statistical analysis Data are presented as mean ± standard deviation. The data for total MMP-9 and active MMP-9 from ex-vivo AVM tissues are presented as a relative expression with control brain samples as 100%. We used ANOVA for comparison, and statistical significance was taken at  P  < .05.
Statistical analysis
Data are presented as mean ± standard deviation. The data for total MMP-9 and active MMP-9 from ex-vivo AVM tissues are presented as a relative expression with control brain samples as 100%. We used ANOVA for comparison, and statistical significance was taken at  P  < .05.PResults Ex-vivo treatment of cultured AVM tissues We collected two AVM tissues for ex-vivo treatment with doxycycline. These patients were not enrolled in the pilot clinical study. AVM-I AVM tissue-I was collected from a 28 y.o. patient with a 29 mm AVM (Spetzler-Martin score 3). The patient had an AVM hemorrhage 236 days prior to the AVM resection. The patient did not receive embolization treatment or radiosurgery. AVM tissue-I was divided into 9 wells and treated with 0, 10, and 100 ng/ml of doxycycline (3 wells for each). Two doxycycline doses were selected to test (1) whether an average doxycycline concentration (10 ng/ml) achieved by standard clinical treatment has any effects on MMP-9 in AVM tissue, and (2) whether a high concentration of doxycycline (100 ng/ml) has any effects of viability of AVM tissues in vitro. There was a gradual increase in LDH release over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability (Figure  1 ). However, there was no difference among different treatment groups. To avoid effects from ongoing cellular death and to ensure tissue viability during the treatment, we chose the first 48 hours to assess the effects of doxycycline on MMP-9 levels in AVM tissues. Figure 1 Tissue viability assessed by LDH (lactate dehydrogenase) release.  There was a gradual increase in LDH release (mean ± SD) over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability. However, there was no difference among different treatment groups. Tissue integrity was further assessed by examining H&E staining of ex-vivo cultured tissues (Figure  2 ). On day 0 (before the treatment), although cutting and mincing of the AVM tissues had caused minor tissue injury and distortion, a majority of blood vessels were intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. Figure 2 H&E staining of ex-vivo cultured AVM tissues.  On day 0 (before the treatment), although cutting and mincing of the AVM tissues appeared to cause minor tissue injury and distortion, a majority of blood vessels was intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 (control vs doxycycline 10 μg/ml: 100 ± 8 vs 48 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 8 vs 59 ± 10%-control, P < .05) (Figure  4A ). In addition, there was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml (control vs doxycycline 10 μg/ml: 100 ± 6 vs 60 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 6 vs 61 ± 9%-control, P < .05). (Figure  4B ) There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. A representative zymogram is shown in Figure  3 . Figure 3 Representative zymogram showing MMP-9 and MMP-2 standard, control AVM tissues, AVM tissues treated with 10 μg/ml doxycycline, and AVM tissues treated with 100 μg/ml doxycycline.  In AVM tissues, there were proteolytic bands corresponding to pro-MMP-9 (≈97 kDa) and active-MMP-9 (≈88 kDa). Figure 4 Active and total MMP-9 levels after 48 hours of ex-vivo doxycycline treatment.  AVM-I:  Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 There was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml.  AVM-II:  Doxycycline 10 μg/ml significantly decreased active and total MMP-9. There was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. (mean ± SD) AVM-II AVM tissue-II was collected from a 63 y.o. patient with a 22 mm AVM (Spetzler-Martin score 2). The patients had a history of AVM hemorrhage 167 days prior to the AVM resection. The patient received an embolization treatment 1 day before the AVM resection. The patient did not receive radiosurgery. Using this tissue, we aimed to study effects of doxycycline at more clinically relevant concentrations of doxycycline (1–10 μg/ml). Therefore, AVM tissue-II was treated with 0, 1, 5, and 10 μg/ml of doxycycline for 48 hours. Since the results from AVM tissue-I showed a relatively wide variation in MMP-9 levels in each well among the same treatment group, we increased wells per treatment group from 3 to 4. There was no difference in tissue viability indicated by LDH release among different treatment groups at 48 hours (data not shown). Similar to the AVM-I, doxycycline 10 μg/ml significantly decreased active and total MMP-9 (active MMP-9: control vs doxycycline 10 μg/ml: 100 ± 54 vs 43 ± 15%-control, P < .05; total MMP-9: control vs doxycycline 10 μg/ml: 100 ± 41 vs 59 ± 21%-control, P < .05) (Figure  4C  & 4D ). In addition, there was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. Pilot clinical trial 14 AVM patients were enrolled. The trial was originally started as a double blinded randomized trial, and 9 patients were randomized. In an effort to improve recruitment for this feasibility study, we converted to an open label drug design. 10 patients received doxycycline, and 4 patients received placebo. Clinical data are shown in Table  1 . Subjects were informed regarding possible side effects including gastrointestinal symptoms, cutaneous photosensitivity, skin pigmentation, teeth discoloration, and vestibular side effects. To monitor compliance and possible side effects, study subjects were interviewed by phone three times during one-week treatment period. One patient had nausea that was relieved by taking food with the drug. Other side effects were not noted. Table 1 Characteristics of AVM patients enrolled in a pilot clinical study to test effects of oral doxycycline treatment on MMP-9 in AVM lesions. Group Age Gender S-M score* AVM size** Draining veins History of hemorrhage Number of embolization treatments Dox 50 M 4 38 Superficial & Deep No 1 Dox 48 F 4 30 Deep Yes 0 Dox 52 M 4 42 Superficial & Deep Yes 2 Dox 43 M 3 29 Superficial & Deep No 1 Dox 23 M 4 34 Superficial & Deep No 2 Dox 28 M 4 40 Superficial Yes 2 Dox 56 M 2 10 Superficial No Dox 41 F 3 30 Superficial No 1 Dox 62 M 2 41 Superficial No 1 Dox 42 M 2 2 Superficial Yes 0 Placebo 53 M 2 28 Superficial & Deep No 1 Placebo 17 M 1 18 Superficial No 1 Placebo 48 F 2 13 Deep No 0 Placebo 22 M 3 19 Deep Yes 0 * Spetzler-Martin score (15), **AVM maximum diameter (10). Dox = doxycycline There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group (total MMP-9: 2.18 ± 1.94 vs 3.26 ± 3.58 ng/100 μg protein, P = .50; active MMP-9: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25) (Figure  5 ). Figure 5 Effects of oral doxycycline treatment on MMP-9 in AVM tissues.  There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group. (mean ± SD)
Results
Ex-vivo treatment of cultured AVM tissues We collected two AVM tissues for ex-vivo treatment with doxycycline. These patients were not enrolled in the pilot clinical study.
Ex-vivo treatment of cultured AVM tissues
We collected two AVM tissues for ex-vivo treatment with doxycycline. These patients were not enrolled in the pilot clinical study.AVM-I AVM tissue-I was collected from a 28 y.o. patient with a 29 mm AVM (Spetzler-Martin score 3). The patient had an AVM hemorrhage 236 days prior to the AVM resection. The patient did not receive embolization treatment or radiosurgery. AVM tissue-I was divided into 9 wells and treated with 0, 10, and 100 ng/ml of doxycycline (3 wells for each). Two doxycycline doses were selected to test (1) whether an average doxycycline concentration (10 ng/ml) achieved by standard clinical treatment has any effects on MMP-9 in AVM tissue, and (2) whether a high concentration of doxycycline (100 ng/ml) has any effects of viability of AVM tissues in vitro. There was a gradual increase in LDH release over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability (Figure  1 ). However, there was no difference among different treatment groups. To avoid effects from ongoing cellular death and to ensure tissue viability during the treatment, we chose the first 48 hours to assess the effects of doxycycline on MMP-9 levels in AVM tissues. Figure 1 Tissue viability assessed by LDH (lactate dehydrogenase) release.  There was a gradual increase in LDH release (mean ± SD) over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability. However, there was no difference among different treatment groups. Tissue integrity was further assessed by examining H&E staining of ex-vivo cultured tissues (Figure  2 ). On day 0 (before the treatment), although cutting and mincing of the AVM tissues had caused minor tissue injury and distortion, a majority of blood vessels were intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. Figure 2 H&E staining of ex-vivo cultured AVM tissues.  On day 0 (before the treatment), although cutting and mincing of the AVM tissues appeared to cause minor tissue injury and distortion, a majority of blood vessels was intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 (control vs doxycycline 10 μg/ml: 100 ± 8 vs 48 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 8 vs 59 ± 10%-control, P < .05) (Figure  4A ). In addition, there was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml (control vs doxycycline 10 μg/ml: 100 ± 6 vs 60 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 6 vs 61 ± 9%-control, P < .05). (Figure  4B ) There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. A representative zymogram is shown in Figure  3 . Figure 3 Representative zymogram showing MMP-9 and MMP-2 standard, control AVM tissues, AVM tissues treated with 10 μg/ml doxycycline, and AVM tissues treated with 100 μg/ml doxycycline.  In AVM tissues, there were proteolytic bands corresponding to pro-MMP-9 (≈97 kDa) and active-MMP-9 (≈88 kDa). Figure 4 Active and total MMP-9 levels after 48 hours of ex-vivo doxycycline treatment.  AVM-I:  Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 There was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml.  AVM-II:  Doxycycline 10 μg/ml significantly decreased active and total MMP-9. There was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. (mean ± SD)
AVM-I
AVM tissue-I was collected from a 28 y.o. patient with a 29 mm AVM (Spetzler-Martin score 3). The patient had an AVM hemorrhage 236 days prior to the AVM resection. The patient did not receive embolization treatment or radiosurgery. AVM tissue-I was divided into 9 wells and treated with 0, 10, and 100 ng/ml of doxycycline (3 wells for each). Two doxycycline doses were selected to test (1) whether an average doxycycline concentration (10 ng/ml) achieved by standard clinical treatment has any effects on MMP-9 in AVM tissue, and (2) whether a high concentration of doxycycline (100 ng/ml) has any effects of viability of AVM tissues in vitro.There was a gradual increase in LDH release over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability (Figure  1 ). However, there was no difference among different treatment groups. To avoid effects from ongoing cellular death and to ensure tissue viability during the treatment, we chose the first 48 hours to assess the effects of doxycycline on MMP-9 levels in AVM tissues.Tissue integrity was further assessed by examining H&E staining of ex-vivo cultured tissues (Figure  2 ). On day 0 (before the treatment), although cutting and mincing of the AVM tissues had caused minor tissue injury and distortion, a majority of blood vessels were intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore.Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 (control vs doxycycline 10 μg/ml: 100 ± 8 vs 48 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 8 vs 59 ± 10%-control, P < .05) (Figure  4A ). In addition, there was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml (control vs doxycycline 10 μg/ml: 100 ± 6 vs 60 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 6 vs 61 ± 9%-control, P < .05). (Figure  4B ) There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. A representative zymogram is shown in Figure  3 .AVM-II AVM tissue-II was collected from a 63 y.o. patient with a 22 mm AVM (Spetzler-Martin score 2). The patients had a history of AVM hemorrhage 167 days prior to the AVM resection. The patient received an embolization treatment 1 day before the AVM resection. The patient did not receive radiosurgery. Using this tissue, we aimed to study effects of doxycycline at more clinically relevant concentrations of doxycycline (1–10 μg/ml). Therefore, AVM tissue-II was treated with 0, 1, 5, and 10 μg/ml of doxycycline for 48 hours. Since the results from AVM tissue-I showed a relatively wide variation in MMP-9 levels in each well among the same treatment group, we increased wells per treatment group from 3 to 4. There was no difference in tissue viability indicated by LDH release among different treatment groups at 48 hours (data not shown). Similar to the AVM-I, doxycycline 10 μg/ml significantly decreased active and total MMP-9 (active MMP-9: control vs doxycycline 10 μg/ml: 100 ± 54 vs 43 ± 15%-control, P < .05; total MMP-9: control vs doxycycline 10 μg/ml: 100 ± 41 vs 59 ± 21%-control, P < .05) (Figure  4C  & 4D ). In addition, there was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9.
AVM-II
AVM tissue-II was collected from a 63 y.o. patient with a 22 mm AVM (Spetzler-Martin score 2). The patients had a history of AVM hemorrhage 167 days prior to the AVM resection. The patient received an embolization treatment 1 day before the AVM resection. The patient did not receive radiosurgery.Using this tissue, we aimed to study effects of doxycycline at more clinically relevant concentrations of doxycycline (1–10 μg/ml). Therefore, AVM tissue-II was treated with 0, 1, 5, and 10 μg/ml of doxycycline for 48 hours. Since the results from AVM tissue-I showed a relatively wide variation in MMP-9 levels in each well among the same treatment group, we increased wells per treatment group from 3 to 4.There was no difference in tissue viability indicated by LDH release among different treatment groups at 48 hours (data not shown). Similar to the AVM-I, doxycycline 10 μg/ml significantly decreased active and total MMP-9 (active MMP-9: control vs doxycycline 10 μg/ml: 100 ± 54 vs 43 ± 15%-control, P < .05; total MMP-9: control vs doxycycline 10 μg/ml: 100 ± 41 vs 59 ± 21%-control, P < .05) (Figure  4C  & 4D ). In addition, there was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9.Pilot clinical trial 14 AVM patients were enrolled. The trial was originally started as a double blinded randomized trial, and 9 patients were randomized. In an effort to improve recruitment for this feasibility study, we converted to an open label drug design. 10 patients received doxycycline, and 4 patients received placebo. Clinical data are shown in Table  1 . Subjects were informed regarding possible side effects including gastrointestinal symptoms, cutaneous photosensitivity, skin pigmentation, teeth discoloration, and vestibular side effects. To monitor compliance and possible side effects, study subjects were interviewed by phone three times during one-week treatment period. One patient had nausea that was relieved by taking food with the drug. Other side effects were not noted. Table 1 Characteristics of AVM patients enrolled in a pilot clinical study to test effects of oral doxycycline treatment on MMP-9 in AVM lesions. Group Age Gender S-M score* AVM size** Draining veins History of hemorrhage Number of embolization treatments Dox 50 M 4 38 Superficial & Deep No 1 Dox 48 F 4 30 Deep Yes 0 Dox 52 M 4 42 Superficial & Deep Yes 2 Dox 43 M 3 29 Superficial & Deep No 1 Dox 23 M 4 34 Superficial & Deep No 2 Dox 28 M 4 40 Superficial Yes 2 Dox 56 M 2 10 Superficial No Dox 41 F 3 30 Superficial No 1 Dox 62 M 2 41 Superficial No 1 Dox 42 M 2 2 Superficial Yes 0 Placebo 53 M 2 28 Superficial & Deep No 1 Placebo 17 M 1 18 Superficial No 1 Placebo 48 F 2 13 Deep No 0 Placebo 22 M 3 19 Deep Yes 0 * Spetzler-Martin score (15), **AVM maximum diameter (10). Dox = doxycycline There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group (total MMP-9: 2.18 ± 1.94 vs 3.26 ± 3.58 ng/100 μg protein, P = .50; active MMP-9: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25) (Figure  5 ). Figure 5 Effects of oral doxycycline treatment on MMP-9 in AVM tissues.  There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group. (mean ± SD)
Pilot clinical trial
14 AVM patients were enrolled. The trial was originally started as a double blinded randomized trial, and 9 patients were randomized. In an effort to improve recruitment for this feasibility study, we converted to an open label drug design.10 patients received doxycycline, and 4 patients received placebo. Clinical data are shown in Table  1 . Subjects were informed regarding possible side effects including gastrointestinal symptoms, cutaneous photosensitivity, skin pigmentation, teeth discoloration, and vestibular side effects. To monitor compliance and possible side effects, study subjects were interviewed by phone three times during one-week treatment period. One patient had nausea that was relieved by taking food with the drug. Other side effects were not noted.Characteristics of AVM patients enrolled in a pilot clinical study to test effects of oral doxycycline treatment on MMP-9 in AVM lesions.* Spetzler-Martin score (15), **AVM maximum diameter (10). Dox = doxycyclineThere was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group (total MMP-9: 2.18 ± 1.94 vs 3.26 ± 3.58 ng/100 μg protein, P = .50; active MMP-9: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25) (Figure  5 ).Discussion In this study, we demonstrated the feasibility of doxycycline, a tetracycline derivative, to decrease MMP-9 activity in AVM tissues. First, we demonstrated that a clinically relevant concentration of doxycycline decreased MMP-9 without affecting tissue viability in ex-vivo AVM tissues. Second, there was a trend that oral administration of doxycycline for as short as one week in AVM patients resulted in a decrease in MMP-9 at the target site – in the AVM nidus. By decreasing MMP-9 activity in AVM tissues, doxycycline may be able to restore the structural stability of AVM blood vessels and modify the clinical course of AVMs. Our data will provide the basis to conduct a clinical study to assess effects of tetracycline derivative treatment on the prevention of hemorrhage from AVMs. MMP-9 is a major enzyme that degrades the vascular extracellular matrix and has been implicated in a number of vascular diseases that involve abnormal angiogenesis and vascular remodeling [ 7 ]. MMPs are reported to be increased in cerebral aneurysms, atherosclerotic carotid plaque, and abdominal aortic aneurysm [ 8 - 10 ]. MMPs are emerging as a potentially new therapeutic target to treat vascular diseases. It has been proposed that pharmacological inhibition of MMPs may stabilize the unstable blood vessels and prevent complications such as vessel rupture [ 7 ]. In patients with abdominal aortic aneurysm, doxycycline treatment for one week prior to the repair surgery resulted in decreased MMP-9 and MMP-2 in the wall of the aneurysms [ 10 ]. Similar results have been reported in patients with atherosclerotic carotid plaques who received doxycycline for 2–8 weeks [ 11 ]. Our data using ex-vivo AVM tissues showed that abnormally high levels of MMP-9 expression in AVM tissues could be reduced by a clinically relevant concentration of doxycycline (i.e. 1–10 μg/ml). In patients with abdominal aortic aneurysm, doxycycline treatment at a conventional dose, 200 mg/day, resulted in a mean plasma concentration of doxycycline at 4.6 μg/ml with a range of 1.3 to 14.4 μg/ml in humans, and there was a corresponding reduction in plasma MMP-9 levels [ 12 ]. Similar plasma levels of doxycycline in mice successfully inhibited growth of experimental abdominal aortic aneurysms [ 13 ]. We were able to show that doxycycline at 10 μg/ml for as short as 48 hours can reduce MMP-9 expression in AVM tissues. A longer duration of treatment often used in clinical settings may result in a more pronounced reduction in MMP-9 expression in AVM tissues. One of the limitations of the ex-vivo study was the relatively short duration of doxycycline treatment. We chose 48 hours treatment to ensure tissue viability and integrity. Although we chose the doxycycline concentrations based on plasma doxycycline levels achieved by a commonly used therapeutic regimen, it is possible that tissue levels of doxycycline might be significantly lower than those of plasma. Furthermore, functional consequences of the reduction of MMP-9 in AVMs need to be examined in animal models or clinical studies. However, at the present time, there is no animal model that approximates recurrent intracranial hemorrhage from AVMs. There are models that can mimic certain aspects of the AVM phenotype. Hyperstimulation of mouse brain with VEGF using adenoviral transduction causes an increase in capillary density, increased MMP-9 activity, and may, in the appropriate genetic background, result in small vascular malformations [ 14 ]. Doxycycline can reduce capillary density and MMP-9 activity in this model [ 15 ]. Doxycycline has been shown in other human vascular diseases to reduce MMP levels. For example, Axisa et al. treated patients undergoing carotid endarterectomy with doxycycline or placebo for 2–8 weeks. Although MMP-1, MMP-3, and MMP-9 were reported to be increased, only MMP-1 had a statistically significant reduction by doxycycline; there was a trend for MMP-9 to be decreased by 22%. Baxter et al. used a much longer treatment period, 6 months, in patients with abdominal aortic aneurysm. They reported a gradual reduction of plasma MMP-9 levels by oral doxycycline over 6 month treatment period (baseline vs 3 months vs 6 months: 119 ± 38 vs 84 ± 33 vs 66 ± 24 ng/ml) [ 12 ]. To further explore the feasibility of a clinical application of doxycycline in modifying the clinical course of AVMs, we conducted a pilot clinical trial. This trial was primarily designed to test the effects of a short-term treatment with oral doxycycline on the expression of MMP-9 in AVM tissues. Despite the small sample size and short duration of treatment used in this study, there was a clear trend for one week of oral doxycycline treatment prior to surgical resection to reduce expression of both active and total MMP-9 in AVM tissues. These results indicate the feasibility of oral treatment with tetracycline derivatives in reducing MMP-9 activity in AVM tissues. Despite a large difference in the mean values of MMP levels in our pilot data, variance of MMP levels was high. Accordingly, a sample size of 46 in each group would be needed to demonstrate a difference in active MMP-9, assuming an alpha of 0.05 and power of 80%. However, variance may be reduced by utilizing a longer duration of treatment, as suggested by studies in other diseases [ 11 , 12 ]. A clinical study with a modestly larger sample size and longer treatment duration should suffice to verify effects of oral doxycycline on reducing MMP-9 activity in AVMs. There are some further considerations that may account for our high variance that could be avoided in future studies. Surgical AVM tissues consist mainly from nidal tissues, but they may contain intervening astroglial or neuronal tissues that may not express angiogenic factors to the same extent as nidal tissue. Histological examination of each AVM tissue fragments to confirm a predominance of nidal tissues may yield more homogenous tissue and decrease variability in MMP-9 levels. Some of the homogenized samples underwent a several "thaw-freeze" processes during the preliminary phase of the experiments, which may have increased variability in MMP-9 levels. Embolization treatment may affect production and activation of MMPs. Our previous study, however, showed no association between MMP-9 and embolization treatment in 37 AVM patients [ 16 ]. Based on the experience from this pilot study, future studies can be performed in a more standardized fashion to decrease tissue-to-tissue variability. We used different methods to measure MMP-9 in the medium collected from ex-vivo AVM tissues and homogenized AVM tissues from the pilot clinical trial. We chose zymography, a gel based method detecting enzymatic activity of MMPs, for ex-vivo AVM tissues, because the medium volume that can be used for MMP-9 assay was extremely limited, and our preliminary experiments showed higher sensitivity of zymography compared to ELISA. One of the disadvantages of using zymography is that "gel-to-gel" variability in sensitivity makes it difficult to combine the data obtained from multiple gels. On the other hand, ELISA method allows researchers to assay approximately 40 samples at a time when duplicates are used. In addition, establishing a standard curve, ELISA method can express MMP-9 expression values by the absolute unit such as μg/ml or ng/100 μg protein. Therefore, for the clinical pilot trial, in order to quantitatively compare MMP-9 expression among a large number of samples originally planned, we used ELISA to MMP-9 expression.
Discussion
In this study, we demonstrated the feasibility of doxycycline, a tetracycline derivative, to decrease MMP-9 activity in AVM tissues. First, we demonstrated that a clinically relevant concentration of doxycycline decreased MMP-9 without affecting tissue viability in ex-vivo AVM tissues. Second, there was a trend that oral administration of doxycycline for as short as one week in AVM patients resulted in a decrease in MMP-9 at the target site – in the AVM nidus. By decreasing MMP-9 activity in AVM tissues, doxycycline may be able to restore the structural stability of AVM blood vessels and modify the clinical course of AVMs. Our data will provide the basis to conduct a clinical study to assess effects of tetracycline derivative treatment on the prevention of hemorrhage from AVMs.MMP-9 is a major enzyme that degrades the vascular extracellular matrix and has been implicated in a number of vascular diseases that involve abnormal angiogenesis and vascular remodeling [ 7 ]. MMPs are reported to be increased in cerebral aneurysms, atherosclerotic carotid plaque, and abdominal aortic aneurysm [ 8 - 10 ]. MMPs are emerging as a potentially new therapeutic target to treat vascular diseases. It has been proposed that pharmacological inhibition of MMPs may stabilize the unstable blood vessels and prevent complications such as vessel rupture [ 7 ]. In patients with abdominal aortic aneurysm, doxycycline treatment for one week prior to the repair surgery resulted in decreased MMP-9 and MMP-2 in the wall of the aneurysms [ 10 ]. Similar results have been reported in patients with atherosclerotic carotid plaques who received doxycycline for 2–8 weeks [ 11 ].Our data using ex-vivo AVM tissues showed that abnormally high levels of MMP-9 expression in AVM tissues could be reduced by a clinically relevant concentration of doxycycline (i.e. 1–10 μg/ml). In patients with abdominal aortic aneurysm, doxycycline treatment at a conventional dose, 200 mg/day, resulted in a mean plasma concentration of doxycycline at 4.6 μg/ml with a range of 1.3 to 14.4 μg/ml in humans, and there was a corresponding reduction in plasma MMP-9 levels [ 12 ]. Similar plasma levels of doxycycline in mice successfully inhibited growth of experimental abdominal aortic aneurysms [ 13 ]. We were able to show that doxycycline at 10 μg/ml for as short as 48 hours can reduce MMP-9 expression in AVM tissues. A longer duration of treatment often used in clinical settings may result in a more pronounced reduction in MMP-9 expression in AVM tissues.One of the limitations of the ex-vivo study was the relatively short duration of doxycycline treatment. We chose 48 hours treatment to ensure tissue viability and integrity. Although we chose the doxycycline concentrations based on plasma doxycycline levels achieved by a commonly used therapeutic regimen, it is possible that tissue levels of doxycycline might be significantly lower than those of plasma. Furthermore, functional consequences of the reduction of MMP-9 in AVMs need to be examined in animal models or clinical studies. However, at the present time, there is no animal model that approximates recurrent intracranial hemorrhage from AVMs. There are models that can mimic certain aspects of the AVM phenotype. Hyperstimulation of mouse brain with VEGF using adenoviral transduction causes an increase in capillary density, increased MMP-9 activity, and may, in the appropriate genetic background, result in small vascular malformations [ 14 ]. Doxycycline can reduce capillary density and MMP-9 activity in this model [ 15 ].Doxycycline has been shown in other human vascular diseases to reduce MMP levels. For example, Axisa et al. treated patients undergoing carotid endarterectomy with doxycycline or placebo for 2–8 weeks. Although MMP-1, MMP-3, and MMP-9 were reported to be increased, only MMP-1 had a statistically significant reduction by doxycycline; there was a trend for MMP-9 to be decreased by 22%. Baxter et al. used a much longer treatment period, 6 months, in patients with abdominal aortic aneurysm. They reported a gradual reduction of plasma MMP-9 levels by oral doxycycline over 6 month treatment period (baseline vs 3 months vs 6 months: 119 ± 38 vs 84 ± 33 vs 66 ± 24 ng/ml) [ 12 ].To further explore the feasibility of a clinical application of doxycycline in modifying the clinical course of AVMs, we conducted a pilot clinical trial. This trial was primarily designed to test the effects of a short-term treatment with oral doxycycline on the expression of MMP-9 in AVM tissues. Despite the small sample size and short duration of treatment used in this study, there was a clear trend for one week of oral doxycycline treatment prior to surgical resection to reduce expression of both active and total MMP-9 in AVM tissues. These results indicate the feasibility of oral treatment with tetracycline derivatives in reducing MMP-9 activity in AVM tissues.Despite a large difference in the mean values of MMP levels in our pilot data, variance of MMP levels was high. Accordingly, a sample size of 46 in each group would be needed to demonstrate a difference in active MMP-9, assuming an alpha of 0.05 and power of 80%. However, variance may be reduced by utilizing a longer duration of treatment, as suggested by studies in other diseases [ 11 , 12 ]. A clinical study with a modestly larger sample size and longer treatment duration should suffice to verify effects of oral doxycycline on reducing MMP-9 activity in AVMs.There are some further considerations that may account for our high variance that could be avoided in future studies. Surgical AVM tissues consist mainly from nidal tissues, but they may contain intervening astroglial or neuronal tissues that may not express angiogenic factors to the same extent as nidal tissue. Histological examination of each AVM tissue fragments to confirm a predominance of nidal tissues may yield more homogenous tissue and decrease variability in MMP-9 levels. Some of the homogenized samples underwent a several "thaw-freeze" processes during the preliminary phase of the experiments, which may have increased variability in MMP-9 levels. Embolization treatment may affect production and activation of MMPs. Our previous study, however, showed no association between MMP-9 and embolization treatment in 37 AVM patients [ 16 ]. Based on the experience from this pilot study, future studies can be performed in a more standardized fashion to decrease tissue-to-tissue variability.We used different methods to measure MMP-9 in the medium collected from ex-vivo AVM tissues and homogenized AVM tissues from the pilot clinical trial. We chose zymography, a gel based method detecting enzymatic activity of MMPs, for ex-vivo AVM tissues, because the medium volume that can be used for MMP-9 assay was extremely limited, and our preliminary experiments showed higher sensitivity of zymography compared to ELISA. One of the disadvantages of using zymography is that "gel-to-gel" variability in sensitivity makes it difficult to combine the data obtained from multiple gels. On the other hand, ELISA method allows researchers to assay approximately 40 samples at a time when duplicates are used. In addition, establishing a standard curve, ELISA method can express MMP-9 expression values by the absolute unit such as μg/ml or ng/100 μg protein. Therefore, for the clinical pilot trial, in order to quantitatively compare MMP-9 expression among a large number of samples originally planned, we used ELISA to MMP-9 expression.Conclusions In summary, results suggest that doxycycline may have a therapeutic potential to reduce MMP-9 activity in AVM tissues and stabilize blood vessels that are prone to rupture. Our findings may provide a basis for a larger clinical trial to study effects of tetracycline derivatives in preventing AVM hemorrhage.
Conclusions
In summary, results suggest that doxycycline may have a therapeutic potential to reduce MMP-9 activity in AVM tissues and stabilize blood vessels that are prone to rupture. Our findings may provide a basis for a larger clinical trial to study effects of tetracycline derivatives in preventing AVM hemorrhage.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions TH, a corresponding author, participated in all aspects of the study and manuscript preparation. MMM participated in study design, experiments, analysis, interpretation of data, and manuscript preparation. JFL participated in experiments, analysis and interpretation of data. MTL participated in study design, analysis, interpretation of data, and manuscript preparation. WLY participated in study design, analysis, interpretation of data, and manuscript preparation.
Authors' contributions
TH, a corresponding author, participated in all aspects of the study and manuscript preparation. MMM participated in study design, experiments, analysis, interpretation of data, and manuscript preparation. JFL participated in experiments, analysis and interpretation of data. MTL participated in study design, analysis, interpretation of data, and manuscript preparation. WLY participated in study design, analysis, interpretation of data, and manuscript preparation.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: